35-7300

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: CRISPR-Cas9 mediated genome editing ofEzrin (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR864181_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of Ezrin was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with Ezrin Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Ezrin Monoclonal Antibody (3C12) (Product # 35-7300) using 1:1,000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:5,000 dilution).Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toEzrin (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Ezrin Monoclonal Antibody (3C12) (Product # 35-7300) and a ~70kDa band corresponding to Ezrin was observed across HeLa, HCT 116, A-431 and HaCaT compared to NTERA and HEK-293. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HCT 116 (Lane 2), A-431 (Lane 3), HaCaT (Lane 4), NTERA-2 cl.D1 (Lane 5), HEK-293 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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 2 Increased MYC and EZR gene expression in the nine CTC lines compared with HT-29 cells. ( A ) Comparison of MYC expression in CTC lines, HT-29, and SW620 cells (microarray data). ( B ) RT-qPCR analysis of MYC expression in each CTC line and in HT-29 and SW620 cells. ( C ) Comparison of EZR expression in CTC lines, HT-29, and SW620 cells (microarray data). ( D ) EZR expression level in each CTC line, HT-29, and SW620 cells. ( E ) Immunofluorescence analysis of ezrin expression in the nine CTC lines. c: p -value = 1.00 x 10 -4 ; d: p -value = 6.41 x 10 -7 . All RT-qPCR results were normalized to the B2M expression level in each sample; * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Enterohemorrhagic Escherichia coli infection caused calpain-dependent loss of mirovillar ezrin and ezrin cleavage . Lysates from subcellular fractions of polarized control CaCo-2a CON and HOX monolayers with and without 6 h EHEC infection by were probed for ezrin by western blot. Asterisk (or arrow) indicates the ~55 kDa ezrin cleavage product (A) . Polarized CaCo-2a CON cells without (B) and with 6 h infection by EHEC (C) and EHEC-infected polarized HOX monolayers (D) were stained for ezrin (green) and EHEC (red). EHEC was detected by anti-O157 antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Protrusions give rise to PS + vesicles containing Lm A. PS + bacteria and ezrin + protrusions were enumerated in HeLa cells infected at an MOI of 100 with wild type Lm for the indicated time. Averages +/- s.d. for 3 independent experiments. B. Cells were transfected with LifeAct-RFP (F-actin probe) and then infected with wild type Lm expressing GFP for 6 h. Live infected cells were analyzed by spinning disk confocal microscopy with Annexin V-Alexa 647 in the medium to label exofacial PS (green). Successive frames are shown. Arrows indicates protrusion that acquires PS. Images representative of 4 independent experiments. Scale bar, 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 InlC is needed for efficient spreading and protrusion formation in a polarized cell line ( a ) InlC promotes spreading. Caco-2 BBE1 cells in transwells were infected with wild-type (wt) or Delta inlC Listeria strains. (i): Representative images of plaque assays. (ii): Average relative plaque diameters or surface areas +- s.d. (n=3). ( b ). InlC does not affect F-actin tail formation. (i). Average percentage (%) +- s.d. (n=3) of intracellular wild-type or Delta inlC bacteria with symmetric F-actin or comet tails. P = 0.45. (ii). Average percentage (%) +- s.d. (n=3) of intracellular bacteria with comet tails only. P = 0.46. ( c ) InlC does not influence tail length. (i). Average tail lengths (mum) +- s.d. (n=3). P = 0.84. (ii). Distributions of tail lengths:

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Increased apoptosis sensitivity and decreased proliferation in PAICS , MALAT1 and MAST2 knockdown cells. A . MelJuSo melanoma cells with stable pGIPZ shRNA-mediated PAICS knockdown (sh1-3 PAICS ) and control shRNA (sh ctr )-transduced cells were incubated for 24 hours with 0.2 uM staurosporine. Apoptosis was quantified by FACS using the Nicoletti protocol [36] , and data are presented as the mean +- SEM, n = 4. Efficient knockdown of PAICS was confirmed in Western blot analysis using an anti-PAICS antiserum (inlet). B . Cell expansion kinetics of MelJuSo cells upon pGIPZ shRNA-mediated PAICS knockdown (sh1-3 PAICS ). Viable cells were quantified using a CASY cell counter, and cell numbers were compared with cells transduced with a non-targeting control shRNA (sh ctr ). Data represent the mean values +- SEM, n = 3. One-way-ANOVA testing with Bonferroni multi-comparison correction was performed. The significance is indicated by stars for the comparison of sh ctr versus sh1-3 PAICS (*: p-value

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Xenograft tumor growth of MAST2 and PAICS knockdown in U87 and MelJuSo cells, respectively. A . The subcutaneous xenograft tumor growth of stable sh2 MAST2 knockdown U87 cells was quantified with a manual caliper 2-3 times per week and compared with non-targeting control shRNA (sh ctr )-transduced cells. Data are presented as the mean +- SEM. 5 mice/group were injected into the right flank. The unpaired, two-tailed t-test was performed individually for each day after injection of the tumor cells and revealed the following p-values: day 23, 0.0063; day 25, 0.0063; day 28, 0.0169; day 30, 0.0127; day 32, 0.0219; day 34, 0.0496. B . pTRIPZ sh2 PAICS -transduced MelJuSo were injected subcutaneously into the right flank of NOD-SCID mice. When the xenografted tumors reached a size of 50 mm 3 , mice received drinking water containing 2 mg/ml doxycycline and 10 g/l sucrose ad libitum until the end of the experiment to induce the PAICS shRNA knockdown. The graph displays the relative tumor growth (tumor volumes from day x/day 0 of doxycycline treatment). Injected mice per group: sh ctr = 10, sh2 PAICS = 11. Data are presented with the mean values and error bars indicate SEM. The unpaired, two-tailed t-test was performed individually for each day after the start of doxycycline treatment and revealed significant differences for day 4 to day 28 with doxycycline treatment (day 4, 0.0028; day 7, 0.0163; day 11, 0.0024; day 14, 0.0082; day 18, 0.0414; day 21, 0.0429; day 25, 0.0311

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2. Smad3-mediated upregulation of phosphorylated ezrin involves HG -induced podocyte apoptosis. (A) Human podocytes were treated with HG (30 mM) for 0, 12, 24 and 48 h, respectively. Mannitol (MN) was used as the osmotic controls. The levels of the phosphorylated ezrin at Thr567 and total ezrin were assessed using western blotting. n =4 independent experiments. Data are presented as mean+-s.d. One-way ANOVA. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3. HG inhibits PKA activation through downregulation of cAMP by the phosphorylated ezrin in podocytes. (A,B) Human podocytes were treated with HG (30 mM) for 0, 12, 24 and 48 h, respectively. Mannitol (MN) was used as the osmotic controls. PKA activity (A) and intracellular cAMP levels (B) were measured and compared. n =4 independent experiments. Data are presented as mean+-s.d. One-way ANOVA. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Increased MYC and EZR gene expression in the nine CTC lines compared with HT-29 cells. ( A ) Comparison of MYC expression in CTC lines, HT-29, and SW620 cells (microarray data). ( B ) RT-qPCR analysis of MYC expression in each CTC line and in HT-29 and SW620 cells. ( C ) Comparison of EZR expression in CTC lines, HT-29, and SW620 cells (microarray data). ( D ) EZR expression level in each CTC line, HT-29, and SW620 cells. ( E ) Immunofluorescence analysis of ezrin expression in the nine CTC lines. c: p -value = 1.00 x 10 -4 ; d: p -value = 6.41 x 10 -7 . All RT-qPCR results were normalized to the B2M expression level in each sample; * p < 0.05, ** p < 0.01, *** p < 0.001.