- PA5-31066 - Invitrogen Antibodies EIF6 antibody

Submitted references Eukaryotic translation initiation factor 6 overexpression plays a major role in the translational control of gallbladder cancer.

Golob-Schwarzl N, Wodlej C, Kleinegger F, Gogg-Kamerer M, Birkl-Toeglhofer AM, Petzold J, Aigelsreiter A, Thalhammer M, Park YN, Haybaeck J
Journal of cancer research and clinical oncology 2019 Nov;145(11):2699-2711

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), K-562 (Lane 3), C2C12 (Lane 4), Hep G2 (Lane 5) and A549 (Lane 6). The blot was probed with Anti-eIF6 Polyclonal Antibody (Product # PA5-31066, 1:5000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 26 kDa band corresponding to eIF6 was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using eIF6 Polyclonal Antibody (Product # PA5-31066). Various whole cell extracts (30 µg) were separated by 12% SDS-PAGE, and the membrane was blotted with eIF6 Polyclonal Antibody (Product # PA5-31066) diluted at 1:5,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of eIF6 was achieved by transfecting HeLa with eIF6 specific siRNAs (Silencer® select Product # s7586 ). Western blot analysis (Fig. a) was performed using whole cell extracts from the eIF6 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with eIF6 Polyclonal Antibody (Product # PA5-31066, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to eIF6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of eIF6 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with eIF6 Rabbit Polyclonal Antibody(Product # PA5-31066) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nucleus, Nucleolus and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry (Paraffin) analysis of eIF6 was performed in paraffin-embedded human colon tissue using eIF6 Polyclonal Antibody (Product # PA5-31066) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 eIF6 expression is increased in GBC compared to NNT. a Representative immunoblots of eIF6 protein expression in fresh frozen GBC samples compared to NNT. b Densitometrical analysis of fresh frozen GBCs ( n = 14) proved the significantly increased expression of eIF6 in tumor tissue compared to NNT (* p < 0.05). The intensity of the bands was normalized to GAPDH, which served as loading control. Due to Gaussian distribution of data, Student's t test was performed for statistical analysis. c qRT-PCR of EIF6 mRNA was performed in fresh frozen 11 GBC and fresh frozen 9 NNT samples. Fold change values of EIF6 normalized to GAPDH as housekeeping gene are depicted. Bars represent mean +- SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis: Mann-Whitney U test. d High expression is highlighted in red and low expression in blue. Kaplan-Meier curves represent the correlation between EIF6 gene expression and survival of BTC patients based on TCGA database an in silico analysis stratified by the median. Statistical analysis: log-rank test ( p = 0.193)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Knockdown of eIF6 in TFK-1 cell line. a Representative immunoblots of successful knockdown of eIF6 with siRNA after 48 and 72 h in TFK-1 cell line. b Densitometrical analysis of eIF6 signals normalized to GAPDH, which served as loading control. In TFK-1 cells, eIF6 protein levels are decreased after 48 and 72 h post-transfection, compared to scrambled siRNA-transfected condition. c mRNA levels of EIF6 in transfected TFK-1 cells analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Three independent experiments were carried out. Bars represent mean +- SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis: one-way ANOVA with Bonferroni post-test. d Representative colony formation assay of eIF6 knockdown induced 48 h and 72 h post-transfection in TFK-1 cell line. e Cell viability of TFK-1 cells transfected with eIF6 siRNA after 48 h and 72 h (*** p < 0.001). f Graphs show apoptosis rates after eIF6 knockdown compared to SC after 48 h and 72 h (** p < 0.001). Three independent experiments were carried out. Bars represent mean +- SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis: two-way ANOVA with Bonferroni post-test

PA5-31066

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