Ab157201

The following primary antibodies were used in WB analysis, rat anti-TGF-β2 (771,244, Novus), rabbit anti-TGF-β1 (ab215715, Abcam), 14–3-3ζ (ab51129, Abcam), C-Raf (ab181115, Abcam), phospho-C-Raf (Ser621) (ab157201, Abcam), phospho-C-Raf (Ser259) (ab173539, Abcam), phospho-MEK1/2 (Ser217/221) (#9154, CST), phospho-ERK1/2 (Thr202/Tyr204) (#9101, CST) and GAPDH (#5174, CST) antibodies. The following secondary antibodies were used in WB, anti-rabbit IgG, HRP-linked Antibody (#7074, CST) and anti-rat IgG, HRP-linked Antibody (#7077, CST).
The following antibodies were used in IHC staining, rabbit anti-Cleaved Caspase-3 (#9661, CST), Cyclin D1 (#55,506, CST), TGF-β1 (ab215715, Abcam) and 14–3-3ζ (ab51129, Abcam) antibodies.
The following primary antibodies were used in the IF, rabbit anti-C-Raf (ab181115, Abcam) antibody and mouse anti-14–3-3ζ (abs100410, Absin, China) antibody. The following secondary antibodies were used in IF, Anti-mouse IgG, Alexa Fluor® 594 Conjugate Antibody (#8890, CST) and Anti-rabbit IgG, Alexa Fluor® 488 Conjugate Antibody (#4412, CST).

To monitor dephosphorylation by PP5, 0.15 μM of HSP90–CDC37–BRAF^V600E complex or HSP90–CDC37–CRAF complex was mixed with 0.3 μM of PP5 in a buffer containing 100 mM NaCl, 25 mM Hepes pH 8, 10 mM KCl, 1 mM MnCl2, 0.2 mM TCEP, 2% glycerol and 2.5 mM MgCl₂. The reaction was started by incubating the samples at 30 °C. Samples were taken over 45 min for SDS-PAGE analysis. Phosphorylation-site specific antibodies were used in western blots to probe the phosphorylation states of phospho-Ser13 Cdc37 (MA533209 Invitrogen) diluted 1/2500, phospho-Ser729 BRAF (ab124794 Abcam) diluted 1/1000 and phospho-Ser621 CRAF (ab157201 Abcam) diluted 1/1000. Uncropped raw images for this and all other western blots presented are shown in Supplementary Fig. 5.

The immunofluorescence analysis was performed as previously described (Huang et al., 2016) with minor modification. Briefly, the epididymal spermatozoa were placed on gelatin‐coated slides, air‐dried, and fixed with ice‐cold methanol for 10 min at −20°C. The resulting slides were blocked with 5% goat serum (BOSTER) for 2 h at room temperature and then incubated with primary antibodies against PSMA3 (bs‐9352R; Bioss Biotechnology Inc.), PSMA3 (phospho S250; bs‐9353R; Bioss Biotechnology Inc.), RAF1 (bs‐1703R; Bioss Biotechnology Inc.), and RAF1 (phospho S621; ab157201; Abcam) overnight at 4°C at a dilution of 1:100. After washing three times with PBS, the slides were incubated with secondary antibody labeled with fluorescein isothiocyanate (ab6717; 1:200; Abcam) for 1 h at room temperature, and then coverslipped in Prolong Gold antifade reagent with 4′,6‐diamidino‐2‐phenylindole (Life Technologies) and kept in the dark until photographed using an Olympus IX73 inverted fluorescence microscope (Olympus). For the negative control, the primary antibody was replaced with normal rabbit IgG.