Ab24550

Whole-cell extracts were prepared from LNCaP, LNCaP-AI, and PC-3 cells in RIPA buffer (50 mM Tris-HCl ph 8.0, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% SDS). For cell fractionation, cells were lysed with cytoplasmic buffer (10 mM HEPES ph 7.6, 50 mM NaCl, 0.5 M sucrose, 1 mM DTT, 5 mM MgCl₂, 0.1% Triton X-100). After centrifugation, the supernatant was the cytoplasmic fraction and the nuclear pellet was washed 3 times with cytoplasmic buffer followed by lysis with RIPA buffer. Extracts were subjected to electrophoresis on SDS/PAGE and then transferred to nitrocellulose membranes for immunoblot analysis as described [33 (link)]. Antibodies for TBLR1#1 (sc-100908) were purchased from Santa Cruz antibodies. Antibodies for H2B and GAPDH were purchased from Cell Signaling. Antibodies for TBLR1#2 (ab24550) and TBLR1#3 (ab84141) were purchased from Abcam.

Colorimetric (DAB, 3,3′-diaminobenzidine) histochemistry was performed as described in Comptour et al. (2014) (link) with minor modifications and using the Novolink polymer detection system (7140-K, Leica Micro-systems). In brief, 4-µm-paraffin sections of testes were dewaxed in xylene and rehydrated in a graded series of alcohol baths. The sections were first incubated for 30 min at 95 °C and then incubated for 10 min at room temperature in 0.01 M sodium citrate solution (pH 6) for antigen retrieval. Slides were washed under running water for 10 min and incubated for 30 min in a blocking solution (1× PBS, 1% BSA, 0.1% Triton). Antibody against TBL1XR1 (ab24550, Abcam) and TBL1X (ab24548, Abcam) was diluted at 1/50. Using a wet chamber, slides were incubated overnight at 4 °C with antibody diluted in blocking buffer. The subsequent stages were performed as described by the manufacturer (Novolink polymer detection, Leica), and the sections were counterstained with Mayer’s hematoxylin solution for 2 min (Merck, Millipore). Sections were dehydrated in a graded series of alcohol bath and a final xylene bath.