rabbit anti eif2α  (Proteintech)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ENDOU-1-induced cytoplasmic HnRNPA3 recognizes m6A methylation on the upstream reading frame of human CHOP transcripts to achieve maximal CHOP translation

doi: 10.1007/s00018-026-06180-7

Figure Lengend Snippet: HnRNPA3 is positively correlated with CHOP translation. A Schematic showing the dual-luc reporter constructs for measuring huORF chop -mediated translational inhibition. phRG-TK was used as an internal control. B Dual-luc assay was used to analyze the effect of HnRNPA3 on h uORF chop -MTI. Histograms present the luc activity obtained from HEK293T cells co-transfected with puORFchop‐luc, phRG‐TK, and each indicated plasmid and then treated with either DMSO (control group; grey column) or Thapsigargin (TH; stress group; solid column) for 6 h, followed by analysis of luc activity. Cells transfected with pCS2vector and kept at normal conditions served as a control group. Relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from the control group normalized to 1. Data were averaged from three independent trials and presented as mean ± SEM. ***P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test.) ( C ) Histograms show the luc activity obtained from zebrafish embryos microinjected simultaneously with puORFchop‐luc, phRG‐TK, and each indicated plasmid, followed by analysis of luc activity at 96 h post-fertilization (hpf). Embryos microinjected with the pCS2 vector during normal conditions (grey column) served as a control group, while the microinjected embryos at 72 hpf subjected to 40°C for 1 h comprised the heat‐shocked stress group (solid column). Relative luc activity was determined as above. *P ≤ 0.001; ***P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test.) ( D ) Western blot analysis. The protein levels of p‐eIF2α, total eIF2α, and CHOP were detected in cells overexpressing protein as indicated under either non-stress (TH, -) or stress (TH, +) conditions. The α‐tubulin and GAPDH served as internal controls. E Using quantitative RT-qPCR to determine the relative expression level of CHOP mRNA in control or HnRNPA3-overexpressing HEK293T cells under either normal (DMSO) or stress (TH) conditions. Data were averaged from three independent trials and presented as mean ± SEM. F Dual-luc assay was used to analyze the effect of HnRNPA3-knockdown on h uORF chop -MTI under either control (DMSO) or stress (TH) conditions in HEK293T cells. Data were averaged from three independent trials and presented as mean ± SEM. Statistical analysis was performed as above. G Western blot analysis. The protein levels of p‐eIF2α, total eIF2α, and CHOP expressed in HnRNPA3-knockdown cells were detected under either non-stress (TH, -) or stress (TH, +) conditions. The α‐tubulin and GAPDH served as internal controls. H RT-qPCR analyses of the gradient distribution of three mRNAs after polysome profiling assay (PPA). Lysates from control (pCS2; blank column), ENDOU-1-overexpressing (ENDOU-1; solid column), and HnRNPA3-overexpressing (HnRNPA3; grey column) cells were subjected to PPA. The resultant fractions from 1‐10 collected from a sucrose gradient were subsequently subjected to RT-qPCR assay to quantify CHOP transcripts ( CHOP mRNAs). The distribution showing the relative abundance of CHOP transcripts contained in each fraction was determined. I Relative abundance (in percentage) of CHOP mRNA presented within monosome- and polysome-containing fractions. Fractions labeled as ‘’untranslated’’ contained 40S, 60S ribosomal subunits (fractions 3 ~ 5), while fractions labeled as ‘’translated’’ contained 80 S monosome, light and heavy polysomes (fractions 6–10). Data were averaged from three independent trials and presented as mean ± SEM. *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test. Protein levels relative to each internal control (α-tubulin or GAPDH) are presented below each lane

Article Snippet: The following antibodies were used: CHOP (1:1000; CST, #2895), HnRNPA3 (1:1000; Proteintech, 25142-1-AP), ATF4 (1:1000; Proteintech, 10835-1-AP), eIF2α (1:1,000; CST, #5324); p-eIF2α (1:1,000; CST, #3398); PERK (1:1,000; CST, #3192); p‐PERK (1:1,000; Abcam, Ab156919 ); Bip (1:1,000; Proteintech, 11587-1-AP); Flag (1:20,000; Proteintech, 20543-1-AP); Myc(1:1,000; CST, #2276); ENDOU‐1 (1:1,000; Abcam, EPR15137 ); m6A (1:1000; Proteintech, 68055-1-Ig), METTL14 (1:1,000; Sigma-Aldrich, HPA038002), WTAP (1:1,000; Proteintech, 10200-1-AP), Lamin B1 (1:1000; Santa Cruz, sc-374015), GAPDH (1:20000; Abcam, Ab181602 ), α‐tubulin (1∶10,000; Sigma-Aldrich, T6074), Multi-rAbTM HRP-Goat Anti-Mouse Recombinant Secondary Antibody (1:20,000; Proteinech, RGAM001) and Multi-rAbTM HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (1:20000; Proteintech, RGAR001).

Techniques: Construct, Inhibition, Control, Activity Assay, Transfection, Plasmid Preparation, Comparison, Western Blot, Quantitative RT-PCR, Expressing, Knockdown, Labeling