Ab57336

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.

Tissue culture cells were fixed in 4% paraformaldehyde or ice-cold 100% methanol for 10 minutes, permeabilised in 0.1% Triton, and blocked in 3% bovine serum albumen (ThermoFisher Scientific). Mouse photoreceptor cells were isolated from retina by gentle dissection before fixation and staining as for tissue culture cells. Antibodies used were as follows: rabbit anti-CROCC (1:250–1:750, Novus Biologicals NBP1-80820), mouse anti-NEDD1 (1:500, Abcam ab57336), rabbit anti-PCNT (1:1000; Abcam ab4448), mouse anti-SAS6 (1:300, Santa Cruz Biotechnology sc-81431), mouse anti-CENPJ (1:100, Santa Cruz Biotechnology sc-81432), mouse anti-gamma Tubulin (1:1000, ice-cold methanol; GTU-88), mouse anti-CETN1 (1:4000, EMD Millipore 20H5), mouse anti-CEP164 (1:200, Santa Cruz Biotechnology sc-515403), rabbit anti-CEP350 (1:500, Atlas Antibodies HPA030845), rabbit anti-CNAP1 (Proteintech, 14498-1-AP), rabbit anti-CDK5RAP2 (Atlas Antibodies, HPA046529), alpaca anti-GFP nanobody (1:400, Chromotek gba-488), and FluoTag-X2 anti-mScarlet (1:500, NanoTag Biotechnologies, N1302-At565).