Anti b raf sc 5284

Anti-dynein intermediate chain (DIC) (mAb 1685) was from Chemicon (Billerica, MA). Anti-phospho-p44/42MAPK (phospho Erk1/2; 4370S), anti-p44/42 MAPK (Erk1/2; 4780), anti-phospho-MEK1/2 (9121S), anti-phospho-JNK (9251S) were from Cell Signaling Technology (Beverly, MA). The mouse anti-Smad2 (610843) and the mouse p150^Glued Ab (610474) were from BD Biosciences Transduction Laboratories (San Jose, CA). Anti-B-Raf (sc-5284) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pan-Ras (MAB5195) was from Millipore (Bedford, MA), whereas anti-RRas (C-8) (SC-166221) was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit monoclonal DYNLL1 Ab (ab51603) was from Abeam (Cambridge, MA). TGFβ1 was purchased from R & D Systems (Minneapolis, MN). Gel filtration standards (151-1901) were purchased from Bio-Rad (Hercules, CA). Our km23-1 antibodies (Abs) (27-43 for Western blotting; 1-15 for immunofluorescence) were produced as previously described (Jin et al., 2007 (link)).

Western blot, immunoprecipitation and in vitro kinase assays were performed as described^{1 (link)}. Antibodies used include: anti-p217/p221-MEK1/2 (p-MEK1/2) (no. 9154), anti-p202/p204-ERK1/2 (p-ERK1/2) (no. 4370), anti-MEK1/2 (no. 4694), anti-ERK1/2 (no. 4696), anti-p-RSK (no. 9346) from Cell Signaling; anti-V5 (R960-25), ThermoFisher Scientific; anti-BRAF (sc-5284), Santa Cruz Biotechnology; anti-Flag (F3165) Sigma; anti-CRAF (610152), BD Transduction Laboratories; anti-p-CRAF S338 (05-538), Millipore; anti-RAS from the active RAS pull-down and detection kit (ThermoFisher Scientific, no. 16117). For immunoprecipitations of tagged proteins we used: anti-V5 agarose affinity gel (Sigma, A7345), anti-Flag M2 affinity gel (Sigma, F1804), protein G agarose gel (ThermoFisher Scientific, no. 15920010).

EBC lysis buffer was prepared to lyse cell as follows: 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM Complete Protease Inhibitors (Roche Diagnostics, Risch-Rotkreuz, Switzerland), and 1 mM Phosphatase Inhibitor Cocktail III (Sigma-Aldrich). The cell lysate was mixed with Laemmli sample buffer then used to perform Western blot analysis following the procedures as described previously (Long et al., 2012 (link)). The following primary antibodies were used: anti-BRAF (sc-5284; Santa Cruz Biotechnology, Dallas, TX), anti-ERK3 (ab53277; Abcam, Cambridge, UK), anti-pERK1/2 (4370; Cell Signaling Technology, Danvers, MA), anti-Rictor (A300-459A-M; Bethyl Laboratories, Montgomery, TX), anti-sin 1 (A300–910A-T; Bethyl Laboratories), anti-β-actin (3700; Cell Signaling Technology), anti-GAPDH (ab181602; Abcam). β-Actin and GAPDH were used as loading control. Western blot imaging was performed and analyzed by Amersham Imager 600 and ImageQuantTL software (GE Healthcare Life Sciences, Chicago, IL).