PA1-16927

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of Min6 cell lysate showing specific labeling of the ~110 kDa Phospho-IRE1 alpha protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Phospho-IRE1 alpha (Ser724) in COS-7 untransfected, COS-7 expressing wild-type IRE1 alpha, COS-7 expressing kinase-dead IRE1 alpha. Samples were incubated in Phospho-IRE1 alpha (Ser724) polyclonal antibody (Product # PA1-16927). Lane 1: COS-7 untransfected Lane 2: COS-7 expressing wild-type IRE1 alpha Lane 3: COS-7 expressing kinase-dead IRE1 alpha. Theoretical molecular weight: 110 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Phospho-IRE1 alpha (Ser724) in HeLa cells. Samples were incubated in Phospho-IRE1 alpha (Ser724) polyclonal antibody (Product # PA1-16927) using a dilution of 2.0 µg/mL followed by an anti-rabbit HRP secondary antibody. Treated (+) or untreated (-) with 10 mM DTT for 60 min to activate the UPR. Total protein was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% BSA in TBST. Detection: chemiluminescence. Theoretical molecular weight: 110 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Phospho-IRE1 alpha (Ser724) in Min6 cells. Samples were incubated in Phospho-IRE1 alpha (Ser724) polyclonal antibody (Product # PA1-16927). Treated with different concentrations of glucose for 3 hours prior to lysates preparation.

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Androgens divergently regulate the UPR arms LNCaP cells were cultured and treated with R1881 for the indicated times. A, B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR (qPCR). Controls were treated with vehicle for 84 h and set to 100. Data represent the mean of three independent experiments in triplicate, and bars represent SE. P -values ranged between 1.66 x 10 -5 and 0.025, and IRE1alpha expression in R1881 48 h was * P = 0.013 with respect to vehicle-treated cells using unpaired Student's t -test. C-E IRE1alpha , XBP-1S , and XBP-1U mRNA in CWR22 xenografts grown in nude mice and collected at the indicated times after castration. The value at t = 0 was set to 1. Columns represent the mean of at least three independent tumors for each time point, and bars represent SE. P -values ranged between 5.7 x 10 -10 and 0.0002. IRE1alpha expression at 48 h post-castration was 3.17 x 10 -6 with respect to t = 0 using unpaired Student's t -test. F, G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis. Data presented are representative of three independent experiments. Source data are available online for this figure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Effects of PDI knockdown on ER stress induction and excitatory receptor expressions. ( A ) Western blot data for ER stress makers including pPERK, pIRE1alpha, IRE1alpha and ATF6 at 7 days after PDI siRNA infusion. M, molecular weight marker. ( B ) Western blot data for excitatory receptors including IP3R, NR2A, NR2B, NR1, M1R and CIB1 at 7 days after PDI siRNA infusion. M, molecular weight marker. ( C ) Quantification of effects of PDI siRNA on levels of ER stress markers and receptor expressions (n = 7, respectively). PDI knockdown does not induce ER stress and alterations in excitatory receptor expressions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 Exogenous expression of TMEM33 in MCF-7 breast cancer cells is associated with constitutive activation of the PERK and IRE1alpha axes of the UPR signaling. MCF-7 cells were transiently transfected with Myc - TMEM33 or pCR3.1 control vector. Adherent and floating cells were collected 24 h post-transfection. Cells were lysed in RIPA lysis buffer supplemented with protease inhibitor (Roche), 10 mM glycerophosphate, 1 mM sodium orthovanadate, 5 mM pyrophosphate, and 1 mM PMSF. Cell lysates (10 mug protein) were immunoblotted with indicated antibodies (all 1:1000 dilution)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Selective elevation of UPR markers with end-stage AD pathology. i) Representative images of western blots probed for a BiP, b p-PERK/PERK, c p-eIF2alpha/eIF2alpha and d p-IRE1alpha/IRE1alpha. Molecular weights and diagnosis of non-AD (C) and AD (A) are indicated, alongside images of Coomassie total protein stain as loading controls. Markers were quantified according to ii) diagnosis and iii) disease severity (low, 0-2; moderate, Braak 3-4; severe, Braak 5-6). Significant post hoc inter-group differences are indicated (*). For all phosphorylated markers, scatter plots are shown as phospho-signal adjusted for total relative to appropriate controls. BiP ( n = 45), p-PERK ( n = 37), p-eIF2alpha ( n = 45), p-IRE1alpha ( n = 23) data displayed as scatter plots with means with 95% confidence intervals, * p < 0.05

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 (a, b) Representative images showing p-PERK and p-IRE1 immunofluorescence in PC12 cells. (c, d) The average IOD of p-PERK and p-IRE1 per cell. Bars = 25 mu m (magnification: x800). The data are shown as the mean +- SEM, ** P < 0.01 compared with the control group; # P < 0.01, ## P < 0.01 compared with the DEX group. PERK: PKR-like endoplasmic reticulum kinase; IRE1: inositol requirement 1; DEX: dexamethasone; 4-PBA: 4-phenylbutyric acid.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Aging induces ER stress in monkey knee cartilages. Articular cartilage sections of young (6-11 yrs, n=3) or old (20-34 yrs, n=3) monkeys were analyzed immunohistochemically for ER stress markers including BIP, P-IRE1alpha, XBP1, ATF4, and CHOP. Images on left panels are low magnification (Scale bars: 100 um). The areas inside the small rectangles were magnified and displayed in right panels (Scale bars: 20 um).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 ( A ) Western blot analysis demonstrates the inhibition of imiquimod-induced phosphorylation of PERK and IRE 1alpha, the expression and phosphorylation of ATF -4 in melanoma cell lines in response to the treatment with 4- PBA , the inhibitor of ER stress. ( B ) Analyses of band intensity on films are presented as the relative ratio of phospho- PERK , phospho- IRE 1alpha, phospho- ATF 4 and ATF 4 to actin. Bars represent mean +- SD from three blots. * P < 0.05 versus control. # P < 0.05 versus 4- PBA , ++ P < 0.05 versus 4- PBA +imiquimod. EMSA demonstrates the inhibition of imiquimod-induced NF -kappaB activation in BLM ( C ) and MV 3 ( D ) cell lines in response to the treatment with 4- PBA . ( E ) MTT assay demonstrates the abrogation of imiquimod-induced death of both BLM and MV 3 cells in response to the treatment with 4- PBA , the inhibitor of ER stress. ( F ) MTT assay demonstrates the inhibition of imiquimod-induced cell death, in part, by the pre-treatment of melanoma cell lines BLM and MV 3 with the inhibitors of PERK ( GSK 2606414), calpain ( ALLM ), ROS ( NAC ) and completely by the combination of the inhibitors of PERK , ROS with the inhibitor of calpain, whereas, the pre-treatment of melanoma cells with inhibitor of NF -kappaB pathway (Bay11-7082) or the knockdown of XIAP by its specific si RNA enhance imiquimod-induced apoptosis. ( G ) MTT assay demonstrated the inhibition of imiquimod-induced cell death, in part, by the pre- treatment of melanoma cell

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 LPS suppresses phospho-IRE1alpha in mice lungs. Western Blot analysis of phosphorylated IRE1alpha (pIRE1alpha) and IRE1alpha in mice lungs treated with either vehicle (saline) or LPS (1.6 mg/kg) via an intratracheal injection for 24 h. The signal intensity of pIRE1alpha and IRE1alpha was analyzed by densitometry. Protein levels were normalized to IRE1alpha. Image J software (NIH) was used to perform densitometry of immunoblots. Graphpad Prism (version 5.01) was used to analyze the data. Student's t-test was performed to determine the statistical differences among groups. **P < 0.01 vs vehicle (VEH), number of animals in each group = 3. Means +- SEM. Fig. 1

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Effects of hsp90 inhibition in major inflammatory cascades of the murine inflamed lungs. Western blot analysis of phosphorylated ATR (pATR) and ATR ( A ), phosphorylated ATM (pATM) and ATM ( B ), phosphorylated AMPK (pAMPK) and AMPK ( C ), phosphorylated IRE1alpha (pIRE1alpha) and IRE1alpha ( E ) expression in lungs retrieved from mice 72 h after intratracheal injection of either vehicle (saline) or LPS (1.6 mg/kg) and post-treated (24 h after LPS) with an intraperitoneal injection of either AUY-922 (10 mg/kg each, dissolved in 10% DMSO) or vehicle (10% DMSO in saline). The signal intensity of protein bands was analyzed by densitometry. Protein levels of pATR, pATM, pAMPK and pIRE1alpha were normalized to ATR, ATM, AMPK and IRE1alpha, respectively. * p < 0.05, ** p < 0.01 vs. vehicle (VEH) and $ p < 0.05, $$ p < 0.01 vs. LPS, n = 3 animals per group. Means +- SEM. Western Blot analysis of phosphorylated IRE1alpha (pIRE1alpha) and IRE1alpha ( D ) in mouse lungs treated with either vehicle (10% DMSO in saline) or AUY-922 (10 mg/kg each, dissolved in 10% DMSO) via an intraperitoneal injection for 48 h. The signal intensity of pIRE1alpha and IRE1alpha was analyzed by densitometry. Protein levels of pIRE1alpha were normalized to IRE1alpha. * p < 0.05 vs. vehicle (VEH), number of animals in each group = 3. Means +- SEM.--indicates the absence, and + indicates the presence of the corresponding reagent in the treatments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 3 Evidence for ER stress in dTHP-1 but not A549 cells. The relative abundance of phosphorylated IRE1 (A, C) and phosphorylated PERK (B, D) in response to 6 h ricin treatment (black bars; 20 ng/ml) or controls (white bars) in dTHP-1 cells. Bars represent the semi-quantitative signal density of the phosphorylated form of each protein relative to the total pool of that protein probed sequentially on the same membrane swatch. Digitized films of representative western blots selected from among three to four replicate experiments. Membrane swatches were probed with anti-phospho-PERK (Cell Signaling Technology Cat# 3179, RRID:AB_2095853) or anti-IRE1 (Cell Signaling Technology Cat# 3294, RRID:AB_823545), then stripped and re-probed with anti-PERK (Cell Signaling Technology Cat# 3192, RRID:AB_2095847) or anti-phospho-IRE (Thermo Fisher Scientific Cat# PA1-16927, RRID:AB_2262241) antibodies. Results at 3 h treatments were variable and thus were not included in the semi-quantitative graphs in (A) and (B). Refer to Figures S3-S6 to observe the original films from which these images were derived

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. MCOLN1/TRPML1 Deficiency Increased Protein Synthesis and Proteotoxic Stress as a Result of mTORC1 Activation (A-C) Relative rate of incorporation of a pulse of OP-Puro in cultured parental cells (P; black) versus MCOLN1 -deficient clones (red, blue, and green) from A375 (A), M214 (B), and M481 (C) treated with DMSO control (solid bars) or 5 nM Torin1 (striped bars). Data represent mean +- SD from two independent experiments with 3 replicate cultures per treatment per clone per experiment. (D-I) Intracellular protein aggregates were visualized by staining with Proteostat dye (D-F) and then quantified (G-I) in parental cells (black) versus MCOLN1 -deficient clones (red) treated with DMSO control (solid bars) or 5 nM Torin1 (striped bars). Data represent mean +- SD from two independent experiments with 3 replicate cultures per treatment per clone per experiment. (J-L) Western blot analysis of BiP, p-eIF2 alpha (S51), total eIF2 alpha , ATF4, CHOP, p-IRE1 alpha (S724), total IRE1 alpha , and beta-actin in cultured parental cells or MCOLN1 -deficient cells from A375 (J), M214 (K), and M481 (L) treated with DMSO (-) or 5 nM Torin1 (+). The blot reflects one representative experiment (of two performed) per melanoma. Statistical significance was assessed using two-way ANOVAs followed by Sidak's and Dunnett's multiple comparisons tests (A-C) or two-way ANOVAs followed by Sidak's multiple comparisons tests (G-I); ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Trifluoperazine promotes mitochondrial and ER coupled stress. ( A ) Flow cytometry analysis of MiaPaCa-2 cells treated with 10 muM TFP for 24 h was done with MITO-ID for analyzing mitochondrial membrane potential. ROS production was detected using MitoSOX Red ( B ) and CellRox Green ( C ) by flow cytometry analysis in TFP and non-treated cells. ( D ) Representative transmission electron microscopy pictures with different magnifications of the MiaPaCa-2 cells after treatment with 10 muM TFP for 24 h are shown. ( E ) Percentage of mitochondria with ER contacts per field (mean of 10 fields) on each condition; not less than 250 mitochondria were counted. ( F ) Flow cytometry analysis with Fluo-4-AM was performed to determine cytosolic calcium concentration. ( G ) Total RNAs were extracted to monitor the mRNA level of genes involved in the mitochondrial dynamic. ( H ) Western blot analysis was performed to evaluated protein levels of expression of Phospho-IRE1alpha, Phospho-eIF2alpha, CHOP, and beta-actin in TFP-treated cells. For each treatment, statistical significance is * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.001 compared with untreated cells (Student's 2-tailed unpaired t -Test). Data represent mean +- SEM, n = 3 (with technical triplicates).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Hepatic cellular stress response after thermal injury. (A) Representative images of Western blot of multiple cellular stress markers in whole liver lysate in different time after burn injury as compared with sham. (B) to (G) Densitometric analysis of the Western blots of phospho-IRE1alpha (B), CHOP (C), phospho-eIF2alpha/eIF2alpha (D), ATF4 (E), BiP (F) and HSP90 (G). Data are presented as means +- SEM. * P < .05 as compared with all the other groups. # P < .05 as compared with Sham. $ P < .05 as compared with PBD42. p p < 0.05 as compared with PBD21, 28 and 42. N = 6 animals per group including non-tamoxifen control, sham and different time-point post-burn

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 PERK-mediated secretion of proteins under hypoxia. a Total PERK, eIF2alpha and P-eIF2alpha expression in HEK293, LN308 and LN229 cell lines under normoxia or hypoxia. b Active form of ATF6alpha (50 kDa band) in LN308, under hypoxia for 24, 48 and 72 h. EEF2 was used as a loading control. c Relative mRNA levels of XBP1s as determined by qRT-PCR in LN308 and LN229 cell lines under hypoxia (48 h). NDRG1 was taken as a positive control for hypoxia induction. EEF2 was used as housekeeping gene. Data are normalized to the respective normoxic conditions and are represented as the mean of three independent experiments +- SEM ( T test: ** p value < 0.01). N normoxia, H hypoxia. d Total XBP1s mRNA transcripts in HEK293 and LN308 cells treated with hypoxia for 24, 48, 72 h. beta-actin was used as a housekeeping gene. e Total P-IRE1alpha species immunoprecipitated using P-IRE1alpha antibody from HEK293 and LN308 cells treated with hypoxia for 48 h. f Volcano plot representing the regulated secretory proteins from LN308 glioblastoma cells under hypoxic conditions for 72 h without (left) and with PERK inhibitor (GSK2606414; right). The data are represented as the mean of three independent replicates. The significant p value cut-off was set at 0.05.