P21 antibody

Cells were treated as indicated and lysed in Laemmli buffer at 95 °C for 5 min. The denatured protein lysates (~20 μl) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins transferred to nitrocellulose membranes. The blots were divided into three to five horizontal strips guided by protein standards stained by Ponceau Red to permit analysis of multiple proteins from the same sample without antibody stripping. Thereafter membranes were immunoblotted, protein targets visualized and their levels quantified as described previously [56 (link)]. The p21 antibody was obtained from BD Biosciences. The antibodies against FGFRs or p53 were purchased from Santa Cruz. FGFR1 antibody (#MAB765) was from R&D Systems. All other antibodies were supplied by Cell Signaling Technology.

Immunofluorescence staining for determining cellular localization of the AhR was performed as described previously [45 (link)]. Western blotting was performed as described previously [46 (link)]. The p21 antibody was from BD Biosciences and was used at a dilution of 1:400.

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.