Rabbit anti pabp antibody

Yap protein levels were determined by western immunoblotting analysis. In brief, proteins isolated from cells (40 μg) were separated by electrophoresis on a 10% SDS polyacrylamide gel. Partitioned proteins were transferred to a nitrocellulose membrane. The membrane was probed with rabbit anti-Yap antibody (Cell Signaling Technology), rabbit anti-PABP antibody (Abcam), rabbit anti-eIF4G antibody (Cell Signaling Technology), mouse anti-eIF4A, anti-eIF4B or anti-eIF4E (Santa Cruz Biotechnology). HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson ImmunoResearch) was used as the secondary antibody. The corresponding protein bands were visualized using enhanced chemiluminescence reagents. The same membranes were re-probed with HRP conjugated GAPDH antibody (Proteintech), mouse anti-alpha tubulin antibody (Santa Cruz Biotechnology) or mouse anti-PCNA (Santa Cruz Biotechnology) to confirm equal loading of proteins for each sample.

HEp-2 cells were grown on glass coverslips, infected for 24 h then fixed with PBS-formaldehyde 4% (v/v) for 10 min at room temperature and permeabilized with PBS-BSA 1% (w/v)-Triton X-100 0.1% (v/v) for 10 min. For immunofluorescence staining, cells were incubated for 1 h with the indicated primary antibodies, and 30 min with the appropriate Alexa Fluor-conjugated secondary antibodies and Hoechst 33342 (1 μg/ml). After washing in PBS, coverslips were mounted in ProLong Diamond antifade reagent (Thermofisher). For PLA experiments, Duolink In Situ kit (Sigma-Aldrich) was used according to the manufacturer’s instructions. After permeabilization and blocking, cells were incubated with a mouse anti-M2-1 antibody and a rabbit anti-PABP antibody (Abcam) 60 min at room temperature in a humidity chamber. The coverslips were then incubated with corresponding secondary antibodies conjugated with PLA probes for 60 min at 37 °C. Ligation and amplification steps were performed as indicated by the manufacturer and coverslips were mounted onto slides. Z-stack image acquisitions of multi-labelled cells were performed under the WLL Leica SP8 microscope.

Tissues or cell samples were prepared by RIPA (Beyotime, China) and protease inhibitor cocktail. Aliquots of the lysates were electrophoresed in 10% polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Bio-Rad, USA). The membrane was blocked with 5% skim milk/TBST (Tris-buffered saline with 0.1% Tween-20) and probed with various antibodies, e.g., rabbit anti-BOULE (1:1000 dilution; Boule anti-serum 101)^{[16 (link)]}, rabbit anti-PUM2 antibody (1:1000 dilution; Cat. No. ab10361, Abcam, USA); rabbit anti-PABP antibody (1:1000 dilution; Cat. No. ab21060, Abcam); rabbit anti-α-tubulin antibody (1:5000 dilution; Cat. No. SC-8035, Santa Cruz Biotechnology, USA); anti-β-actin (1:1000 dilution; Cat. No. SC-1615, Santa Cruz Biotechnology); mouse anti-FLAG antibody (1:2000 dilution; Cat. No. abF1804, Sigma). Detection of HRP conjugated secondary antibody was performed with enhanced chemiluminescence detection reagents (ECL Kit; PerkinElmer, USA).