γ tubulin sc 7396

Mouse monoclonal antibodies: acetylated Tubulin (T7451, IF: 1:10,000; Sigma-Aldrich), ARL13B (66739-1-Ig, IF: 1:300; Proteintech, 75-287, IF: 1:1,000; NeuroMab), and S-tag (MAC112, IF: 1:100, WB: 1:5,000; EMD Millipore). Rat monoclonal antibody: HA (7c9, WB: 1:1,000; ChromoTek). Rabbit polyclonal antibodies: EGFP (50403-2-AP, IF: 1:200, WB: 1:1,000; Proteintech), Flag (F7425, WB: 1:1,000; Sigma-Aldrich), Adenylyl cyclase III (C-20, IF: 1:100; Santa Cruz), B9D1 (NBP2-84489, WB: 1:1,000; Novus Biologicals) and HTR6 (31 (link)). Goat polyclonal antibodies: γ-Tubulin (sc-7396, IF: 1:200; Santa Cruz). Alexa Fluor (AF)–conjugated donkey secondary antibodies from Thermo Fisher Scientific (all used for IF at 1:10,000): AF488 anti-mouse IgG (A21202), AF488 anti-rabbit IgG (A21206), AF555 anti-mouse IgG (A31570), AF555 anti-rabbit IgG (A31572), AF594 anti-rabbit IgG (A21207), and AF647 anti-goat IgG (A21447). Also from Thermo Fisher Scientific were HRP-conjugated secondary antibodies (used for WB at 62 ng/ml): goat anti-rabbit IgG (A16104), goat anti-mouse IgG (A16072) and donkey anti-rat IgG (A18739). Biotinylated proteins were detected with Neutravidin-HRP (A2664, 1 μg/ml; Thermo Fisher Scientific). D-biotin was from Thermo Fisher Scientific (BP-232-1).

The CEP215 and pericentrin antibodies have been previously described [4] (link), [33] (link). Antibodies specific for NEDD1 (ab57336; Abcam), CEP192 (A302–324A; Bethyl Laboratories), γ-tubulin (sc-7396; Santa Cruz Biotechnology), FLAG (F3165; Sigma-Aldrich), FLAG (#2368; Cell Signaling), α-tubulin (ab18251; Abcam), NuMA (#NA09L; Calbiochem) and γ-tubulin (GTU-88; Sigma-Aldrich) were commercially purchased. Alexa Fluor 488-, 555- and 594-conjugated secondary antibodies (Invitrogen) were used for immunostaining. Anti-mouse IgG-HRP (A9044; Sigma-Aldrich) and anti-rabbit IgG-HRP (AP132P; Millipore) were used as secondary antibodies for immunoblotting.
The NCBI reference numbers of the human CEP215 and pericentrin cDNA sequences are NM_018249.5 and NM_006031.5, respectively. The CEP215^WT and CEP215^ΔC constructs were subcloned into the p3×FLAG-CMV10 vector. The PACT domain used in this study is the C-terminal region of the human pericentrin cDNA (9337–10011 bp). The wild-type and mutant pericentrin constructs (PCNT, PCNT^1235,1241AA and PCNT^{Δ2390–2406}) were subcloned into the p3×FLAG-CMV10-GFP-myc vector. The coding sequences of CEP215 and pericentrin were silently mutated to become resistant to the corresponding siRNAs.