β actin antibody

The fresh tissues were homogenized in a RIPA buffer containing 1 mM PMSF. Equivalent amounts of protein (30 μg) were subjected to Western blot analysis. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and immunoblotted with the indicated antibodies as follows: E-Cadherin antibody (Cat. No. A20798, ABclonal, Wuhan, China), N-Cadherin antibody (Cat. No. A19083, ABclonal, China) and β-actin antibody (Cat. No. K200058M, Solarbio, Beijing, China). After being incubated overnight at 4 °C, the membranes were washed and incubated with the secondary antibody (Cat. No. ZB-2301, ZSGB-Bio, China and Cat. No. ZB-2305, ZSGB-Bio, Beijing, China). The washed membranes were visualized using a chemiluminescence reagent (Millipore, Billerica, MA, USA) and exposed to X-ray film. Data were quantitated using the Image J Software.

Following treatments, harvested cells or colonic tissue samples used for western blotting were prepared using a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5 mM MgCl₂, 10% glycerol, and 1 mM PMSF). Protein contents were obtained post centrifugation (12,000 g for 10 min at 4°C), and their respective concentrations were measured using bicinchoninic acid assay (BCA; Beyotime Biotechnology, Beijing, China) following the instructions of manufacturer. Protein samples were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene difluoride membranes. Non-specific bindings were blocked using 5% nonfat milk at 37°C for about 1 h. Then, the membranes were incubated with the primary antibodies, followed by incubation with peroxidase-conjugated secondary antibody at 37°C for 1 h. Finally, the bands were visualized using enhanced chemiluminescence kit (Beyotime Biotechnology, Beijing, China). The antibodies used in the western blotting assays were shown as follows: phospho-IκBα (Ser36) antibody (Abcam, ab133462), IκBα antibody (Abcam, ab32518), β-actin antibody (ABclonal Technology, AC004), occludin antibody (ABclonal Technolog, A2601), and Horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibodies (DingGuo, Beijing, China).

Total protein from 293T cells was extracted with RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA). Protein concentrations were measured using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). The same amount of protein samples (35 μg) was loaded onto an SDS-polyacrylamide electrophoresis gels and then transferred onto polyvinylidene difluoride membranes (Merck, Billerica, MA, USA). After blocked in 5% skimmed milk for 2 h, the membranes were incubated with primary BCL11A antibody (Abcam, Cambridge, MA, USA; ab19489; 1:1000) overnight at 4°C and with HRP-conjugated rabbit anti-mouse secondary antibody for 60 min at room temperature. The β-actin antibody (Abclonal, Wuhan, China; AC004; 1:1000) was used as internal control. The blots were detected using ECL chemiluminescent reagent (Beyotime Biotechnology, Shanghai, China).

Protein homogenates were made from the left kidney or the cultured TKPTS cells. Western blot analyses were conducted following the manufacturer’s instructions with specific primary antibodies: fibronectin (15613-1-AP, Proteintech, Rosemont, IL, USA), College 1 (14695-1-AP, Proteintech), α-SMA (ab5694, Abcam), tyrosine hydroxylase (TH, ab112, Abcam), p53 (A3185, ABclonal Technology, China), p21 (14-6715-81, Invitrogen), γ-H2AX (#9718, Cell Signaling Technology, Danvers, MA, USA), α_2A-AR (DF3076, Affinity Biosciences), β-arrestin2 (10171-1-AP, Proteintech), VDAC1 (55259-1-AP, Proteintech), COX IV (11242-1-AP, Proteintech), and NF-κB p65 (sc-8008, Santa Cruz Biotechnology). Anti-GAPDH (AC001, ABclonal Technology) or β-actin antibody (AC026, ABclonal Technology) was adopted for loading controls on membranes. Subsequently, the horseradish peroxidase (HRP)-conjugated secondary antibodies (Servicebio, China) were applied to combine the primary ones for 90 min at room temperature. Finally, the ECL system was used to visualize proteins, and the Image J software (NIH, USA) was applied to analyze bands.

Plasmids encoding STAT1 and PLZF-Flag proteins were transiently transfected into HEK-293T cells, and 48 h after transfection, cells were lysed in IP lysis buffer with protease inhibitor cocktail and phenylmethanesulfonyl fluoride (PMSF). Co-IP assays were performed with Flag beeds (Sigma-Aldrich; M8823). For normal western blot, total protein was extracted from GBC cells using RIPA lysis buffer supplemented with 1% PMSF and proteinase inhibitor cocktail. Bicinchoninic acid (BCA) assay was used to measure the protein concentration. Equal amounts of protein were loaded on a 8% sodium salt -polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to NC membranes (Millipore, Bedford, MA). Then, the blots were blocked in 5% skimmed milk with 0.1% Tween 20 for 1 h at room temperature followed by incubating at 4° C overnight with primary antibodies. The membranes were washed with tris-buffered saline and tween (TBST) and then incubated with secondary antibody at room temperature for 2 h. The blots were detected by ECL chemiluminescence kit (Millipore). The PLZF antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). β-Actin antibody from Abclonal Biotech was used as loading control. Other antibodies were purchased from Proteintech group (Proteintech, USA).

Human rhabdomyosarcoma (RD) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 1 μg/ml streptomycin at 37°C in presence of 5% CO₂. The EV71 strain was isolated from patient at Xi’an municipal CDC, propagated and expanded in RD cells. This viral stock was used as parental virus. GuHCl was purchased from Sigma-Aldrich and ribavirin was from MedChemExpress. EV71 VP1 and 3AB antibody were purchased from GeneTex. dsRNA antibody (J2) was from English and Scientific Consulting, Hungary. PI4KB antibody was from Proteintech Group and SCARB2 antibody was from Santa Cruz Biotechnology. β-actin antibody was from ABclonal Technology. DAPI, Alexa Fluor conjugated secondary antibodies for microscopy experiments were purchased from Thermo Fisher Scientific.