Hrp goat anti mouse igg

The protein expression levels were determined by western blot analysis as previously described [36 (link)]. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40 and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary anti-SERPINB2 (Abcam, MA, USA, ab47742) and β-actin (Abcam, MA, USA, ab189073) antibodies overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554021) and HRP goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using an ECL reagent.

Protein extraction and western blot analyses were performed as described previously [19 (link)]. Bax (sc-526), Bcl-2 (sc-492), ATG12 C6 (sc-271688), and GAPDH (sc-32233) antibodies and a mouse anti-goat IgG-HRP secondary antibody (sc-2354) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), NIX from Sigma-Aldrich (Madrid, Spain), and HRP goat anti-mouse IgG from BD Pharmingen™ (San Jose, CA, USA). The proteins were visualized using a Western Lightning Plus-ECL chemiluminescence kit (PerkinElmer, Billerica, MA, USA) according to the manufacturer's protocol. Images were analyzed using the Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Protein band intensity was normalized to GAPDH, and data were expressed in percentage terms relative to controls.

Protein expression levels were determined by western blot analysis as previously described. [56 (link)] Briefly, cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as standard. Samples containing equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-RAD Laboratories). The membranes were blocked with 5% skim milk in Tris buffered saline containing Tween-20 at RT, and the membranes were with primary anti-β-actin (Abcam, Cambridge, MA, USA, ab189073), Oct4 (Abcam, Cambridge, MA, USA, ab19857), Klf4 (Abcam, Cambridge, MA, USA, ab72543), Nanog (Abcam, Cambridge, MA, USA, ab21603), Sox2 (Abcam, Cambridge, MA, USA, ab184149), Wnt1 (Abcam, Cambridge, MA, USA, ab15251), TCF4 (Abcam, Cambridge, MA, USA, ab72586), and LEF1 (Cell Signaling Technology, Beverly, MA, USA, 2230S) antibodies overnight at 4°C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554021) and HRP goat anti-mouse IgG (BD Pharmingen, San Diego, CA, USA, 554002) secondary antibodies for 90 min at RT. Antibody-bound proteins were detected using an ECL.