Anti beclin1 11306 1 ap

The cells were washed with ice-pre-cooled PBS and added with 200 uL RIPA lysate (AWB0136, Abiowell). The total protein was extracted after ultrasonic crushing for 1.5 min. Protein concentrations were determined using the BCA method. Protein samples (200 μg) were separated by SDS-PAGE (12%, AWT0047, Abiowell). Then, proteins were transferred to a polyvinylidene difluoride membrane, which was activated with methanol and blocked with skim milk powder (5%, AWB0004, Abiowell). Membrane was incubated with antibodies, which were included anti-progesterone receptor A (PR-A, ab32085, 1:5000, Abcam, UK), anti-LC3II/I (#43566, 1:1000, CST, USA), anti-Beclin1 (11306-1-AP, 1:1000, Proteintech, USA), anti-p62 (18420-1AP, 1:4000, Proteintech, USA), anti-CytC (10993-1-AP, 1:4000, Proteintech, USA) and anti-β-actin (66009-1-Ig, 1:5000, Proteintech, USA). Then, it was incubated with secondary anti-IgG (SA00001-1/2, 1:6000/5000, Proteintech, USA) at 37°C for 90 min. ECL Plus hypersensitive luminescence solution (AWB0005, Abiowell) was used for visualization, and software (ChemiScope6100, CLINX) was used for imaging analysis.

Antibodies used in these experiments included the following: anti-RND2 (13844–1-AP, Proteintech, USA), anti-Rho7/Rnd2 (GXT56070, GeneTex, USA), anti-p-p38 (#4511, Cell Signaling Technology, USA), anti-p38 (#9212, Cell Signaling Technology, USA), anti-cleaved-caspase3 (ab32042, Abcam, UK), anti-caspase3 (NB100-56708SS, Novus, USA), anti-BAX (50599–2-Ig, Proteintech, USA), anti-GAPDH (#5174, Cell Signaling Technology), anti-P62 (M162–3, Medical Biological Laboratories, Japan), anti-Beclin1 (11306–1-AP, Proteintech, USA), anti-LC3B (GB11124, Servicebio, China), anti-DYKDDDK/Flag-tag (ANT102, Antgene, China), and anti-His-tag (D291–3, Medical Biological Laboratories, Japan). The autophagy inhibitor wortmannin (3-MA) and the autophagy activator rapamycin (Sirolimus) (S1039, USA) were purchased from Selleck (S2758, USA).