Anti gapdh antibody

H9 cells cultured overnight in RPMI1640 medium/0.1% BSA were treated with CCL19 or CCL21 at 37 °C, and then washed with media containing ice-cold PhosStop (Roche, Basal, Switzerland). The cells were lysed by adding 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton-X, PhosStop and Protease Inhibitor Tablets (Sigma). The samples were separated by SDS-PAGE, and transferred onto PVDF membranes for immunoblotting with an anti-phospho-Akt, phospho-Erk1/2, total-Akt, total-Erk1/2 antibody (Cell Signaling Inc., MA, USA) or anti-GAPDH antibody (Biolegend). Bound antibodies were detected using the ECL system (GE Healthcare Life Sciences).

Cells were washed twice with PBS and scraped into ice-cold NP40 cell lysis buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 15,000 rpm for 20 min at 4°C. The protein concentration was determined using the BCA assay. Proteins were separated on SDS/15% polyacrylamide gels, transferred onto nitrocellulose membranes (Thermo Fisher Scientific, Switzerland) utilizing a semi-dry system (Bio-Rad, Switzerland). The membranes were blocked in 5% non-fat milk (50 mg/ml) in TBST, and incubated with the following primary antibodies: anti-p-S129 antibody (Proteintech, 1:1,000, United States), anti-GAPDH antibody (BioLegend, 1:1,000, United States), anti-CK2α antibody (Proteintech, 1:1,000, United States), anti-CK2β antibody (Proteintech, 1:1,000, United States), anti-AcH3K9 antibody (Cell Signaling Technology, 1:1,000, United States), anti-AcH4K5 antibody (Cell Signaling Technology, 1:1,000, United States), anti-Histone 3 antibody (Proteintech, 1:1,000), and anti-Histone 4 antibody (Proteintech, 1:1,000) overnight at 4°C. The membranes were washed three times in TBST and labeled with horseradish peroxidase (HRP)-conjugated secondary antibodies. The signals were developed by enhanced chemiluminescent (ECL). ImageJ software was used to measure the intensity of Western blot bands as described previously (Jensen, 2013 (link)).

Western blotting was done as previously described^{19 (link)}. Total proteins (30 μg/well) were extracted from HAP stem cell colonies cultured from fresh and cryopreserved hair follicles. Sodium dodecylsulfate polyacrylamide (4~20%) was used to perform electrophoresis. A immobilon-P membrane (Millipore Corporation) was used to transfer proteins. Specific protein on Immobilon-P were detected by rabbit polyclonal anti-nestin antibody (1:100), rabbit polyclonal anti-Oct4 antibody (1:500, Abcam), and rabbit polyclonal anti-GAPDH antibody (1:4000, BioLegend). The secondary antibody was peroxidase-conjugated protein A/G (Pierce). An enhanced chemiluminescence (ECL), together with a Western Blotting Detection System (Amersham Biosciences), were used to detect protein. ECL was measured by a Light-Capture CCD Camera System (ATTO, Tokyo, Japan).