Plzf antibody

The following primary antibodies were used: REGγ antibody (Invitrogen, catalog no. 38–3900), p21 antibody (BD Pharmingen, catalog no. 556430), β-actin antibody (Sigma, catalog no. A5316), PLZF antibody (Santa Cruz, catalog no. sc-28319), p53 antibody (Novocastra Laboratories, NCL-p53-CM5p), SCP3 antibody (Abcam, catalog no. ab15093), MVH antibody (Abcam, catalog no. ab13840). C18-4 is a spermatogonial stem cell line with wild-type p53. H1299 is a lung cancer cell line without endogenous p53. A459 is a lung cancer cell line with wild-type p53. GC-1 is a spermatogonial-derived cell line. All the cells are from ATCC and cultured following ATCC standard protocols. The plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen), and the construction of these plasmids is described in the related Experimental Procedures. Cell lysates were collected for protein examination 2 days after transfection. MG132 (10 μM) or Nutlin-3 (10 μM) was used to treat cells for 6 h, and 10 mg/kg of cisplatin was used to treat the mice for 24 h before the sacrifice.

Plasmids encoding STAT1 and PLZF-Flag proteins were transiently transfected into HEK-293T cells, and 48 h after transfection, cells were lysed in IP lysis buffer with protease inhibitor cocktail and phenylmethanesulfonyl fluoride (PMSF). Co-IP assays were performed with Flag beeds (Sigma-Aldrich; M8823). For normal western blot, total protein was extracted from GBC cells using RIPA lysis buffer supplemented with 1% PMSF and proteinase inhibitor cocktail. Bicinchoninic acid (BCA) assay was used to measure the protein concentration. Equal amounts of protein were loaded on a 8% sodium salt -polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to NC membranes (Millipore, Bedford, MA). Then, the blots were blocked in 5% skimmed milk with 0.1% Tween 20 for 1 h at room temperature followed by incubating at 4° C overnight with primary antibodies. The membranes were washed with tris-buffered saline and tween (TBST) and then incubated with secondary antibody at room temperature for 2 h. The blots were detected by ECL chemiluminescence kit (Millipore). The PLZF antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). β-Actin antibody from Abclonal Biotech was used as loading control. Other antibodies were purchased from Proteintech group (Proteintech, USA).

Fifteen micrograms of total proteins were applied to each lane and analyzed by Western blotting. PLZF antibody purchased from Santa Cruz Biotechnology (Dallas, TX, USA) were diluted to 1:200 for the assay. CML antibody purchased from Cosmo Bio (Tokyo, Japan) were diluted to 1:200 for the assay. BSA antibody purchased from Abcam (Cambridge, MA, USA) were diluted to 1:2000 for the assay. Other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and diluted to 1:1000 for the assay. Data of protein bands on Western blots were also quantitated with QuantiScan software (Biosoft, Cambridge, UK).