Anti actin 66009 1 ig

The protein samples were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany), then blocked with 5% bovine serum albumin (BSA, Gbcbio, China). Membranes were probed with anti-p-MLC (#3674T, 1:1000, CST, USA), anti-MLC (#3672S, 1:1000, CST, USA), anti-p-pkcα (#sc-377565, 1:500, SantaCruz, USA), anti-pkcα (#2056S, 1:1000, CST, USA), anti-MLCK (#BM4290, 1:400, BOSTER, China), anti-Calmodulin (#bs-3666R, 1:500, Bioss, China), and anti-actin (#66009-1-Ig, 1:6000, Proteintech, China), followed by horseradish-peroxi-dast-conjugated goat anti-rabbit IgG secondary antibody (#SA00001-2, 1:6000, Proteintech, China) or horseradish-peroxi-dast-conjugated goat anti-mouse IgG secondary antibody (#SA00001-1, 1:6000, Proteintech, China) for 1 h. The blots were detected using an ECL chemiluminescence substrates kit (#WBKLS0100, Millirore, USA).

The antibodies used in this study were as follows: anti-Cdh1 (sc-56312, 1:1000 WB, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-γH2AX (05-636, 1:500 IF, 1:1000 IF, Millipore, Billerica, MA, USA); anti-BRCA1 (sc-6954, 1:200 IF, Santa Cruz); anti-Phospho-Chk1(Ser345) (2348S, 1:1000 WB, Cell Signaling, Danvers, MA, USA); anti-cleaved Caspase-3 (9664S, 1:1000 WB, Cell Signaling); anti-cleaved PARP (9664S, 1:1000 WB, Cell Signaling); anti-Actin (66009-1-Ig, 1:5000 WB, Proteintech, Wuhan, China); anti-GAPDH (60004-1-1g, 1:5000 WB, Proteintech, Wuhan, China), and anti-PICH(8886S, 1:200 IF, Cell Signaling); the secondary antibodies conjugated to horseradish peroxidase were used for Western blotting. The secondary antibodies of anti-mouse, or anti-rabbit containg Alexa Fluor 488 or 594 were used for immunofluorescence staining (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Typically, 2.75 × 10⁵ Oli-neuM cells were seeded in 6-well plates in GM media, and cells were grown to 70% confluence. Treatments were performed for 48 h in DM. For immunoblot analyses, the following antibodies diluted in TBS and 4% BSA were used: anti-actin (66009-1-Ig, 1:5000; Proteintech Group Inc., Rosemont, IL, USA); AbD Serotec (Bio-Rad Laboratories, Hercules, CA, USA): anti-MBP (MCA409S, 1:200). Cell extract preparation and immunoblot analyses were performed as previously described [28 (link),29 (link)].