Sc 789

Cells were prepared for immunostaining as described (Xaver et al. 2013 (link)) with the following modifications. Cells were fixed with three successive incubations (10–15 min, room temperature) in 3.4% formaldehyde, the latter two in 0.1 M potassium phosphate, 0.5 mM MgCl₂, pH 6.4, and then stored at 4°. Spheroplasting used 0.5 mg/ml Zymolyase 100T (Nacalai USA #07655) in place of Zymolyase 20T. Slides were immunostained overnight at 4° or 4 hr at 30° with a mixture of the two primary antisera diluted in blocking buffer [rat anti-tubulin (ab6160 1:1250; Abcam) and rabbit anti-MYC (sc-789 1:250; Santa Cruz)], washed in PBS (three times, 5 min, room temperature), and then incubated with secondary antisera [Cy3-conjugated donkey anti-rabbit IgG (#711-165-152; Jackson Laboratories) and FITC-conjugated rabbit anti-rat IgG (# F1763; Sigma), both 1:600 in blocking buffer] for 3hr at 30°, followed by three 5-min room temperature washes in PBS. Samples to be examined by DAPI-staining only were treated as described (Goyon and Lichten 1993 (link)) after formaldehyde fixation and storage as above.

or western blotting of proteins encoded in the 16p11.2 region, cortical lysates were prepared by solubilizing in cold RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, + protease inhibitors added fresh) for 1 h prior to SDS-PAGE and western blotting with anti-SEZ6L2 (Abcam, ab197058, 1:500), anti-PRRT2 (Sigma, HPA014447, 1:2000), anti-ERK1 (Santa Cruz, sc94, 1:5000), anti-TAOK2 (Santa Cruz, sc-47447, 1:500), anti-FLAG (Sigma, F1804, 1:1000), anti-T7 (Millipore, AB3790, 1:1000), anti-Myc (Santa Cruz, sc-789, 1:1000) antibodies

Embryos were co-injected at one-cell stage with Flag-smad1, HA-foxd4l1.1 and Myc-xbra mRNA constructs in four different groups. The injected embryos were collected at stage 11.5. They were then homogenized in lysis IP buffer. The composition of the IP buffer is described in Kumar et al. (2018)^{15 (link)}. Cell lysates were cleared by centrifugation and were then incubated with Flag-Smad1 (F-2574, Sigma) monoclonal antibody and α-HA (SC-805, Santa Cruz Biotechnology) polyclonal antibody overnight at 4 °C with the immunocomplexes precipitated by protein A/G beads (SC-2003, Santa Cruz Biotechnology). Proper amounts of precipitated beads-protein complex were boiled in the sample buffer, and resolved by electrophoresis in 10% SDS–polyacrylamide gels. Western blotting of Myc-Xbra (SC-789, Santa Cruz Biotechnology) was performed by using an anti-Myc and secondary antibody anti-mouse (SAB-100, Stressgen, Victoria, BC). Immune complexes were visualized by using an ECL detection kit (GE Healthcare).

Specified antibodies to the following proteins were used for immunoblotting (IB), immunoprecipitation (IP), immunofluorescence (IF), and immunohistochemistry (IHC): phospho-Akt1(S473) (1:2000, IB; ab81283, abcam), phospho-Bad(S112) (1:1000, IB, no. 5284, Cell Signaling), phospho-BIM(S69) (1:400, IB; no. 4581, Cell Signaling), phospho-Elk1 (S383) (1:1000, IB; no. 9181, Cell Signaling), phospho-Elk1 (S383) (1:1000, IB; ab34270, abcam), phospho-ERK1/2(TEY) (1:1000, IB; no. 9101L, Cell Signaling), ERK1/2 (1:1000, IB; no. 9102, Cell Signaling), phospho-ERK1/2(T188) (1:1000, IB; 1:500 IHC; Lorenz et al., 2009 (ref. ^{13 (link)}) or A010-40AP, Badrilla), Flag (1:10,000, IB or 1:200, IF; IP 5.6μg/sample;; F3165, Sigma), phospho-Foxo3a(S294) (1:1000, IB; no. 5538, Cell Signaling), Gβ (1:5000, IB; sc-378, Santa Cruz Biotechnology), HA (1:5000, IB; IP 5μg/sample; MMS-101R, Covance), HA (1:500, IF; no. 3724, Cell Signaling), Max (1:200, IF; sc-197, Santa Cruz Biotechnology), c-Myc (1:1000, IB; sc-789, Santa Cruz Biotechnology), c-Myc (1:350, IF; M4439, Sigma), phospho-p70(S6) (1:1000, IB; no. 9204, Cell Signaling), and phospho-p90RSK(S380) (1:5000, IB; ab32203, abcam).