Rabbit anti gapdh

Proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. Nonspecific proteins were blocked with 5% nonfat dried milk in Tris-buffered saline Tween (TBST) buffer for 1 h at room temperature with agitation and then membranes were incubated with various primary antibodies. These were monoclonal mouse anti-Cx43 (1:1000; Invitrogen, Carlsbad, CA), monoclonal rabbit anti-Akt (1:1000; Cell Signaling Technology, Beverly, MA), phosphorylated Akt⁴⁷³ (1:1000; Cell Signaling Technology), or rabbit anti-GAPDH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4–8 °C. After washing 3 times for 10 min per times in TBST, each membrane was incubated with the homologous secondary antibody for 1 h at room temperature. The images were analyzed using Quantity One software (Bio-Rad, Hercules, CA) to obtain the integrated intensities. Each experiment was repeated five times.

Western blot evaluations were undertaken as previously described32 (link). Total protein was extracted from the SOL, EDL and GAS muscles of ground squirrels and solubilized in a sample buffer (100 mM Tris, pH 6.8, 5% 2-β-mercaptoethanol, 5% glycerol, 4% SDS, and bromophenol blue), with muscle protein extracts resolved by SDS-PAGE using Laemmli gels (10% gel with an acrylamide/bisacrylamide ratio of 37.5:1 for Bcl-2 and GAPDH; and 12% gel with an acrylamide/bisacrylamide ratio of 29:1 for Bax and cytochrome C. After electrophoresis, the proteins were electrically transferred to PVDF membranes (0.45μm pore size) using a Bio-Rad semi-dry transfer apparatus. The blotted membranes were blocked with 1% BSA in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris–HCl, pH 7.5) and incubated with rabbit anti-Bcl-2 (50E3) mAb, rabbit anti-Bax large subunit and rabbit anti-cytochrome C (1:1000, Cell Signaling Technology CST, Danvers, MA, USA) and rabbit anti-GAPDH (1:7500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS containing 0.1% BSA at 4 °C overnight. The membranes were then incubated with IRDye 800 CW goat-anti rabbit secondary antibodies (1:5000) for 90 min at room temperature, and visualized with an Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). Quantification analysis of the blots was performed using NIH Image J software.

The collected protein was firstly separated using SDS-PAGE electrophoresis on the basis of size, followed by moving to PVDF membranes with appropriate size. The blot was then immediately placed in 5% fresh non-fat milk powder for blocking for 1 h. Next, the appropriate primary and secondary antibodies were used to incubate the transferred membrane according to the recommended dilution and time in datasheet. The rabbit anti-DNMT3b (1:500), rabbit anti-DNMT3a (1:500), and rabbit anti-histone H3 (1:1,000) were purchased from Cell Signaling Technology (Danvers, MA, USA). The mouse anti-HuR (1:500) and rabbit anti-GAPDH (1:1,000) were purchased from Santa Cruz company (Dallas, TX, USA). Membranes were visualized by the Clarity Western ECL Substrate (Cat. No: 170-5060, Bio-Rad Laboratories, Inc., Hercules, CA, USA), exposed by ChemiDoc Touch (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed by Image J.

Western blot was carried out using the protocol described previously [31 (link)–33 (link)]. The following primary antibodies were used: rabbit anti-XPC (Santa Cruz, USA), rabbit anti-ERK2 (Santa Cruz, USA), rabbit anti-p-ERK1/2 (Santa Cruz, USA), rabbit anti-Snail (Santa Cruz, USA), anti-E-cadherin (Santa Cruz, USA) and rabbit anti-GAPDH (Santa Cruz, USA).

At different time points after infection, cell lysates were prepared by the addition of NP40 lysis buffer (Invitrogen) and protease inhibitor (Roche). Equal aliquots of lysates were heat denatured and separated on a 4–12% SDS-PAGE gel (Invitrogen). Separated proteins were transferred to a methanol-activated polyvinylidine difluoride (PVDF) membrane (Bio-Rad) and blocked in PBS containing 5% skim milk and 0.1% Tween 20. The blot was incubated with mouse anti-N (6B3, in-house), anti-NSs (8G4, in-house), and rabbit anti-GAPDH (Santa Cruz Biotechnology) monoclonal antibodies. The antigen-primary antibody complexes were recognized by alkaline phosphatase (AP)-conjugated goat anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch) antibodies, as appropriate. Visualization of detected proteins was performed by leveraging the colorimetric detection of AP activity using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) substrate.