13-1600

antibody from Invitrogen Antibodies

Targeting: UBC

Provider product page for 13-1600

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [92]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [3]
Other assay [46]

Submit

Product number: 13-1600 - Provider product page
Provider: Invitrogen Antibodies
Product name: Ubiquitin Monoclonal Antibody (Ubi-1)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: 13-1600 recognizes ubiquitin, both conjugated and unconjugated. It reacts with a single chain 8.5 kDa protein. The ubiquitin molecule appears to be present in all eukaryotic cells and has an identical primary structure in all animals. Ubiquitin is present in the nucleus, cytoplasm, and on cell surface membranes.
Antibody clone number: Ubi-1
Concentration: 0.5 mg/mL

UBC protein structure - 13-1600 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Validation of SMIP001 and SMIP004 . (a) Chemical structure of the two small molecule inhibitors of p27 depletions (SMIPs) identified in the screen. (b) Cell lysates from LNCaP-S14 treated with 40 muM SMIPs for 24 h were analysed by immunoblotting. Roscovitine (R) was used as positive control. The data shown is representative of two independent experiments. (c) LNCaP-S14 cells were treated with increasing concentrations of SMIPs (5-40 muM) for 18 h followed by fixation and staining for p27. The graph represents data from 24 replicate wells per concentration. (d) LNCaP-S14 cells were treated with SMIPs (5-40 muM) for 18 h and analysed by immunoblotting for the levels of p27 and p21. (e) p27, p21 and SKP2 stability in LNCaP-S14 cells treated with 40 muM SMIPs for 18 h. Cycloheximide (CHX, 100 mug/mL) was added for the indicated time points prior to preparation of cell lysates and immunoblot analysis. MG132 was used as positive control. The graphs represent the quantification of p27, p21 and SKP2 levels relative to the zero hour time point. The data is representative of three independent experiments. (f) LNCaP-S14 cells were treated with SMIP00s (40 muM) for 18 h followed by extraction of total RNA. The mRNA levels of p27, p21 and SKP2 were quantified by quantitative polymerase chain reaction as described in the Methods section. All data was normalized to GAPDH and is expressed as fold induction. The graph is representative of two independent experiments. (g) Total lysat

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 p300 localizes in aggresomes. A-C . Cos7 cells were treated with DMSO (A) or treated with 5 uM MG132 for 16 hours (B-C) and cells were stained with a monoclonal mix of anti-ubiquitin (A and B), the anti-p300 polyclonal C20 antibody (A-C), or anti-vimentin monoclonal (C) antibodies (see materials and methods for details on the antibodies). The arrow in panel A, indicates the presence of p300 in diffused cytoplasmic aggregates. The arrows in B and C indicate the position of representative aggresomes, enclosed by vimentin and containing ubiquitin. Cells were subjected to fluorescence deconvolution and image reconstruction by using a Zeiss microscope (Axiovert 200M) with deconvolution capabilities (Axiovision 4.1). The merge panels represent the deconvoluted images from the Alexa-Fluor 488 (green), Alexa-Fluor 568 (red), and DAPI signals. D. Quantification of the immuno-fluorescence experiments. The percentage of cells containing aggresomes (black bars) and of aggresomes containing p300 (gray bars) was calculated in mock treated (-) or MG132 treated (+) cells. E. Cos7 cells were mock treated (-; lanes 1 and 4) or treated with MG132 (+; lanes 2, 3 and 5) and cell extracts were subjected to immuno-precipitation with a control antibody (lane 3) or with the anti-p300 antibody (lanes 4 and 5). Immuno-precipitation reactions were divided into different aliquots and subjected to immuno-blot with anti p300 (top panel), anti-HDAC6 (mid panel) or anti dynein antibodies (bottom pan

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 p300 co-localizes with a-synuclein, ubiquitin and HDAC6 in brain of patients affected by Parkinson Disease. Sections of midbrains or cortex from normal control patients ( A ), or from patients by Parkinson Disease ( B-E ) were immuno-stained with the anti-p300 (red) and alpha-synuclein (green, panels B,C ), or with anti-ubiquitin (green, panel D), or with anti-HDAC6 (green, panel E) antibodies as indicated at the top of each panel. Different sections from the same patients or from different patients were subjected to analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 The p300 PSPD is sufficient for aggresome localization. A-D. Cell transfected with the vector expressing RFP alone and treated with MG132 (panel A), or transfected with the RFP-PSPD (panels B-D) were mock treated (B), or treated with MG132 ( C-D ) and subsequently stained for vimentin (green). Panels C and D contain two representative fields of cells transfected with RFP-PSPD. Arrows indicate the position of aggregates formed in the absence (panel B) or in the presence of MG132 (C and D). E. Interaction of p300 with ubiquitinated protein species. The epitope-flagged vector expressing the PSPD (lanes 1-5), or the TAZ2 (lanes 6,7 and 10,11) or the ZZ (lanes 8,9 and 12,13) were transfected in Cos7 cells. Cells were left untreated (-) or treated with MG132 (+), cell extracts were immuno-precipitated with the anti-Flag antibody (lanes 1,2; and 6-to-9), or with a control isotype matched antibody (lane 3) and the products of these immuno-precipitation reactions were probed with ubiquitin antibodies as indicated at the bottom of each panel. Alternatively, the anti-Flag immuno-blot on total cell extracts shows the total amount of p300 proteins present in these reactions, which were derived from approximately 1/50 of total extracts.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8 Effects of p300 on the misfolded protein response. A. A549 cells were transfected with scramble siRNA or with the p300 specific siRNA as described previously, and stained with the anti-ubiquitin (green) or anti-vimentin (red) antibody. The percentage of cells containing ubiquitin aggregates is quantified in panel B . C . A549 cells were transfected with the scramble- or p300-specific siRNAs were mock treated or treated with MG132 for 16 hours, then harvested for flow cytometry. Panel C shows the Propidium Iodide profiles, and percentages of cells in the G1 or G2 phases of the cell cycle are indicated at the top of each relative peak. D. A549 cells transfected with scramble (black bars) or with the p300 siRNAs (gray bars), were treated with MG132. In one set of samples MG132 was washed out after 12-14 hours of treatment (indicated as MG132 w/o) and cells were allowed to recover for about four days, at which time they were counted. Alternatively, cell growth was monitored for the same period of time in the presence of MG132 (indicated as MG132 on). Error bars represent standard deviations. E-F. Control (wt) or p300 mouse embryo fibroblasts (MEF) were mock treated or treated with MG132 for 16-24 hours (panel E), or alternatively, subjected to Heat Shock (HS, panel F) by incubating the cells at 40degC for two hours. Cells were allowed to recover from MG132 treatment or HS for 24-48 hours and were counted. Cell viability was assessed with trypan blue exclusion.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Loss of CSN6 leads to destabilization of Myc and decreases Myc transcriptional activity (a) Immunoblot analysis of Csn6 +/- MEFs. Primary MEF cells were prepared from 13.5-day embryos derived from wild-type (wt) Csn6 +/+ and Csn6 +/- mice. After 4 days of culture, cell lysates were analyzed by indicated antibodies. (b) Quantitative RT-PCR analysis of mRNA levels for indicated c-Myc target genes in CSN6-transfected U2OS cells or Csn6 +/- MEFs. The quantitated mRNA expression level was normalized to GAPDH mRNA. Two MEFs were examined for each genotype. Expression level of transfected CSN6 is indicated as numbers on the heat map, and the heat map depicts the natural logarithm of fold-change in mRNA expression. (c) CSN6 knockdown abrogated serum-induced elevation of Myc. Csn6 +/+ MEFs and Csn6 +/- clone 1 (C1) and clone 2 (C2) MEFs were serum-starved for 24 hours and then were refed with serum for 24 hours. Lysates were analyzed by indicated antibodies. (d) CSN6 decreased Myc turnover. 293T cells were transfected with Flag-CSN6 and were then treated with cycloheximide (CHX; 100 ug/ml) for the indicated times. The immunoblot of Myc signal at each time point was measured using a densitometer and the integrated optical density of Myc was measured. The turnover of Myc is indicated graphically. (e) CSN6 expression affected Myc turnover. 35 S-pulse-labeled HA-Myc protein was immunoprecipitated from indicated transfected 293T cell lysates. The mixture was separated by sodium do

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 14-3-3sigma inhibits cancer metabolism by promoting Myc degradation (a) Loss of 14-3-3sigma upregulated Myc-induced glycolytic genes. The expression of glycolytic genes in HCT116 WT (14-3-3sigma +/+ ) and HCT116 14-3-3sigma -/- cells were measured using Realtime PCR (left panel). Average+-95%CI, n=4, ANOVA,* P< 0.05. Western Blot was performed using the indicated antibodies (right panel). (b) 14-3-3sigma downregulated Myc-induced glycolytic genes. The mRNA expression of glycolytic genes were compared between indicated cells using Realtime PCR (left panel). Average+-95%CI, n=3, ANOVA,* P< 0.05. Western Blot was performed using the indicated antibodies (right panel). (c) 14-3-3sigma reduced expression of Myc-induced glutaminolytic genes. Realtime PCR and Western Blot were used to compare the expression of Myc-induced glutaminolytic genes at mRNA and protein levels. Average+-95%CI, n=3, ANOVA,* P< 0.05. (d) 14-3-3sigma suppressed TFAM expression and reduced MT-CO1/PPRC1 ratio in vitro. TFAM is an important Myc-induced mitochondrial transcription factor. MT-CO1 is a mitochondrial gene encoding cytochrome c oxidase. PPRC1 , peroxisome proliferator-activated receptor gamma, coactivator-related 1, is a gene of nuclear DNA. Average+-95% CI, n=3, Student t-test, * p < 0.05. (e) 14-3-3sigma increased Myc ubiquitination. Indicated cells were treated with Doxycycline to induce Flag-14-3-3sigma expression (+). Non-induced cells were used as a control (-). Cell lysates were immuno

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5. Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H 2 O 2 . (A) PC12 cells were grown in exosome-depleted medium in the presence of H 2 O 2 for 0 min (lanes 1,5), 15 min (lanes 2,6), 1 h (lanes 3,7), or 2 h (lanes 4,8). ECM-bound proteins were released by EGTA (lanes 1-4), whereas extracellular vesicles were isolated from the culture medium by the ExoQuick-TC method (lanes 5-8). 100 ug of protein from the EGTA-released fractions (lanes 1-4) and the control extracellular vesicle fraction (lane 5), or an equal volume of extracellular vesicles from H 2 O 2 -treated cells (lanes 6-8) were separated by 10% SDS-PAGE, transferred to nitro-cellulose membranes and probed with antibodies against pTyr23AnxA2, total AnxA2, CD63, TSG-101 and T-cadherin, as indicated. (B) Following 1 h treatment of PC12 cells with 1 mM H 2 O 2 , proteins (600 ug) present in purified extracellular vesicles were immunoprecipitated (IP) by monoclonal AnxA2 antibodies (lane 1) after pre-clearance of the samples with normal mouse IgG (lane 2). The proteins were subjected to 10% SDS-PAGE and immunoblot analysis using monoclonal antibodies against pTyr23AnxA2 or ubiquitin by loading half of the immunoprecipitation sample on each gel. The bands representing ubiquitylated AnxA2 (square bracket; Ub-AnxA2) and IgG light chain (L C , arrowhead) are indicated to the right. (A,B) Following inc

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Acute genetic deletion of Mule induces concentric hypertrophy with cardiac dysfunction and premature death. ( A ) Down-regulation of Mule mRNA levels in human end-stage heart failure determined by RT- qPCR. ( B ) To specifically inactivate Mule and Myc in the adult heart, we employed a tamoxifen (Tam) inducible Cre- loxP system in which Cre recombinase expression is controlled by the cardiomyocyte-specific myosin heavy chain 6 promoter ( mcm ). 4-hydroxytamoxifen (Tam) was injected intraperitoneally daily for four consecutive days in 10 week-old mice. The day of the last Tam injection was set as day zero. Veh, vehicle. Nt, no treatment. Immunoblot analysis of cytoplasmic Mule and nuclear Myc levels in left ventricular extracts (60 mug total protein/lane) of Mule fl / fl ( y ) ;mcm mice at 7 days post-Tam employing specific antibodies as indicated on the left. Animals were 12 weeks old at the time of analysis. For normalization, Western blots were probed with anti-tubulin (Tub) for cytoplasmic fractions, and anti-nucleophosmin (Npm1) for nuclear fractions. Immunoblots were repeated at least once with similar results. Transcript levels of Mule and Myc in Mule fl / fl ( y ) ;mcm mice at 7 d post-Tam as analyzed by RT-qPCR. n = 4. ( C ) Mule is indispensable for Myc protein stability in the heart by regulation of its ubiquitin-mediated proteasomal degradation. At 7 d post-Tam. Mule fl / fl ( y ) ;mcm , mice were intraperitoneally injected with the proteasomal inhibitor M

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Pup2 predominantly affects CytoQC compared to ERAD. (A) Rescue of the CytoQC-defective phenotype observed with the exogenous expression of wild-type Pup2 ( PUP2 ) in the pup2 -10 mutant. Equal numbers of each strain were spotted as described in Figure 1C with SC-Trp-His and SC-Trp-Ura plates for selection. Vector: empty vector. (B) Missense mutation in spontaneous mutant pup2 -10 is present at residue 101, replacing Leucine for Proline. (C-D) Degradation kinetics of CytoQC and ERAD substrates were determined by pulse chase analyses. Strains were pulsed with 35S-Met/Cys for 5 min for Ste6 *C and Ste6 * and 10 min for ssPrA, CPY* and Sec61 -2, followed by chase for the indicated time points. (E) CytoQC substrate ssPrA is localized predominantly in the nucleus in WT and pup2 -10 . Substrates were detected with anti-HA antibody (green). The ER and nuclear envelope were visualized with anti- Kar2 antiserum (red). Nucleus was visualized with DAPI staining. Scale bar: 2 µm. (F) Accumulation of polyubiquitinated Ste6 *C and DeltassPrA was observed in pup2 -10 mutant compared to WT. Misfolded cytosolic substrates expressed in WT and pup2 -10 were immunoprecipitated (IP) by anti-HA antibody, resolved by SDS-PAGE and analyzed by immunoblotting (IB) with anti-ubiquitin antibody to detect polyubiquitinated substrates.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Reduced free ubiquitin levels cannot fully account for degradation delays in doa4 . (A) Immunoblot of free ubiquitin levels in WT and doa4 deletion strains without any substrate, expressing CytoQC substrates Ste6 *C or ssPrA, and expressing either substrate in the presence of ubiquitin overexpression (Ub). Free ubiquitin was probed with the mouse anti-ubiquitin antibody and PGK was probed as loading control. Unique ubiquitin conjugates characteristic of doa4 are indicated by the black bracket. Graph shows average relative levels of free ubiquitin in doa4 compared to the corresponding WT. Error bars indicate standard deviation of the mean of three independent experiments. (B) Partial restoration of polyubiquitinated substrate levels in doa4 with the overexpression of ubiquitin (Ub). Misfolded CytoQC substrates expressed in WT and doa4 were processed and analyzed as described in Figure 2F . (C) Degradation delay of Ste6 *C and ssPrA was only partially suppressed with ubiquitin overexpression. Strains were pulsed with 35S-Met/Cys as described in Figure 2C . Vector: empty vector.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 SPTBN1 induces ubiquitination and degradation of p65. (A and B) Binding of p65 with IkappaBa kept identical in HCC cells upon the inhibition of SPTBN1. SNU-449 cells were transiently transfected with siRNAs as indicated for 48 h (A) and PLC/PRF/5 cells stably transduced with SPTBN1 shRNA or non-specific (ns) shRNA were cultured for 48h (B). Cells were then subjected to immunoprecipitation (IP) and immunoblotting with antibodies as indicated. Whole cell lysates of above cells (inputs) were analyzed by Western blotting by antibodies as indicated. Data are representative of two to three independent experiments with similar results. The intensities of SPTBN1, p65, IkappaBalpha, pIkappaBalpha and actin were measured by ImageJ software, and the relative expression level of each protein/actin was generated. (C) PLC/PRF/5 cells were transiently transfected as indicated for 48h. Cells were then treated with cycloheximide (CHX) for the indicated time periods and were analyzed for the stability of p65 by Western blotting (upper). The intensities of p65 and actin were measured by ImageJ software, and the relative expression units of p65/actin were generated and normalized relative to the expression unit in 0 h. The percentage remaining of p65 expression (compare to zero hour) was then calculated (lower). (D) SNU-449 cells were transfected as indicated for 48 h. Cells were then treated without or with 10 uM MG132 for 4 h before cell lysis and analyzed by Western blotting. (E) SNU

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 SPTBN1 enhances the expression of SOCS1, which was required for the regulation of p65 by SPTBN1. (A-B) PLC/PRF/5 (A) or SNU-449 (B) cells were transiently transfected with siRNAs as indicated. Cells were then subjected to Western blot analysis. The relative intensities of SOCS1 to actin in two to three independent Western blotting were analyzed as in Figure 2 A and 2 B. Significance of the difference was evaluated using the Student''s t test (* P < 0.05; ** P < 0.01). (C-D) Liver tissues from two pairs of age- and gender-matched WT and Sptbn1 +/- mice were analyzed by Western blotting (C). Liver tissues from two pairs of age- and gender-matched WT and Sptbn1 +/- mice were lyzed and fractionated into cytoplasmic [C] and nuclear [N] fractions, and subcellular distribution of p65 and SOCS1 in each fraction was assessed by Western blotting (D). The relative intensities of p65 and SOCS1 were analyzed as in Figure 2 A and 2 B. (E) SNU-449 cells were transiently transfected as indicated for 48 h and were then analyzed by Western blot analysis. (F) SNU-449 cells were transiently transfected with control siRNA or siRNA to SOCS1 together with empty vector or SPTBN1 plasmid for 48 h. Cells were then analyzed by Western blot analysis. (G) SNU-449 cells were transiently transfected with control siRNA or siRNA to SOCS1 together with empty vector or SPTBN1 plasmid for 48 h. Cells were then treated with 10uM MG132 for 4 h before cell lysis. Cells were immunoprecipitated with antibod

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 PAH content in liver lysates of homozygous and heterozygous Pah-R261Q mice . a Western blots for immunodetection of PAH (alpha-PAH) ( a ) and ubiquitinated protein (alpha-Ub) ( b ) showing the decrease in non-ubiquitinated PAH (non-Ub-PAH; ~51 kDa band) and increase of mono-ubiquitinated PAH (mono-Ub-PAH; ~56 kDa) from genotype Pah WT/WT to Pah R261Q/WT to Pah R261Q/R261Q . The blots are representative from n = 3 replicates for each mice group. GAPDH was used as loading control. c Overview of relative PAH specific activity normalized to activity in Pah WT/WT liver lysates (23.2 +- 2.4 nmol L-Tyr/min/mg protein) ( n = 4 mice for each genotype) and non-Ub-PAH protein (51 kDa) levels from densitometric analysis normalized to both Pah WT/WT liver lysates as well as to GAPDH loading control ( n = 3 mice for each genotype). Data are presented as mean +- SD for Pah WT/WT (purple), Pah R261Q/WT (green), and Pah R261Q/R261Q (ochre), individual values are represented as circles. Differences between genotypes were analyzed by one-way ANOVA followed by Tukey test; differences in PAH activity, p = 0.0005 (***) for Pah WT/WT vs. Pah R261Q/WT , p < 0.0001 (****) for both Pah WT/WT vs. Pah R261Q/R261Q and Pah R261Q/WT vs. Pah R261Q/R261Q ; differences in PAH level, p = 0.0080 (**) for Pah WT/WT vs . Pah R261Q/R261Q . Source data are provided as a Source Data file.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. UBA6 participates in monoubiquitination of LC3B. ( A ) WT and UBA6-KO H4 cells were incubated with 0, 20 or 50 muM MG132 for 6 hr, and analyzed by SDS-PAGE and immunoblotting with antibodies to LC3B, Ub and beta-tubulin (loading control). ( B ) The ratio of LC3B-I to beta-tubulin was determined from experiments such as that in A. The ratio in WT H4 cells without MG132 treatment was arbitrarily set at 1. Bars represent the mean +- SEM of the ratio from three independent experiments. The indicated p -values were calculated using two-way ANOVA with Tukey's multiple comparisons tests. ( C ) WT and UBA6-KO cells were transfected with plasmids encoding HA-Ub and FLAG-LC3B. Cell lysates were immunoprecipitated (IP) with antibody to the FLAG epitope, and cell lysates and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with antibodies to the HA and FLAG epitopes. Ubiquitinated FLAG-LC3B can be seen as a faint band on the long exposure of the anti-FLAG blot (arrow). ( D ) H4 cells were transfected with plasmids encoding HA-Ub and FLAG-LC3B. Cells were incubated with 20 muM MG132 for 6 hr before lysis and immunoprecipitation with antibody to the FLAG epitope. Cell lysates and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with antibodies to the HA and FLAG epitopes. ( E ) Immunofluorescence microscopy showing the localization of FLAG-LC3B and FLAG-LC3B-G120A mutant in WT H4 cells. DAPI (blue) was used to stain the nucleus. Cell edges are outlin