Arl13b 17711 1 ap

The following antibodies were used in the study: acetylated α-tubulin (6–11-B-1, used at 1:10,000 to detect cilia and 1:1,000 to detect cytoplasmic microtubules) and α-tubulin (T5168, used at 1:1,000 for immunofluorescence (IF) and 1:50,000 for western blot) from Sigma; EB1 (610534, used at 1:200), β-catenin (610153, used at 1:200) and anti-PKAc (610980, used at 1:200) from BD biosciences; acetylated α-tubulin (ab24610, used at 1:500) and α-tubulin (ab18251, used at 1:500) from Abcam; IFT54 (HPA037858, Atlas Antibodies, used at 1:50 for IF and 1:1,000 for WB); ZO1 (61–7300, used at 1:100) from Life Technologies; ARL13B (17711-1-AP, Proteintech, used at 1:400); Gp135 (AF1556, R&D, used at 1:200 for IF and 1:1000 for WB); γ-tubulin (DQ-19, Sigma, used at 1:500); γ-tubulin (C-20, used at 1:200), MAP4 (H-300 and G-10, used at 1:400 for IF and 1:1,000 for WB) and ACIII (C-20, used at 1:200) from Santa Cruz; GAPDH (MAB374, used at 1:4000 for WB) from Millipore. Highly cross adsorbed secondary antibodies (Alexa Fluor 488, Alexa Fluor 546, AlexaFluor 555, AlexaFluor 532 and Alexa Fluor 647) were obtained from Molecular Probes (Life Technologies) and were used at 1:200 dilution.

Cells were fixed with cold methanol for 5 min. VPS39-silenced cells were fixed 96 h after the silencing while VPS39-overexpressing cells were fixed 72 h after transfection, blocking and permeabilization were achieved in 0.2% Triton X-100, 10% FBS in PBS. For LC3 detection blocking/permeabilization solution was performed with 0.5% bovine serum albumin (BSA), 0.05% saponin, 50 nM NH4Cl, 0.02% NaN3, in PBS 1X, pH 7.2–7.4). Primary antibodies were: ARL13b (17711-1-AP, Proteintech 1:1000), γ-tubulin (GTU-88, Sigma-Aldrich 1:5000), FLAG-M2 (F3165, Sigma-Aldrich 1:250), IFT20 (13615-1-AP, Proteintech 1:100), OFD1 (HPA031103, Sigma-Aldrich 1:800), LC3B (NB100-2220, Novus Biologicals 1:400). Secondary antibodies Alexa Fluor® IgG were from Thermo Fisher Scientific (1:800). Hoechst 33342 (14 522, Sigma-Aldrich 1:1000) was used to stain nuclei. Confocal fluorescence microscopy and image processing were performed, as described in (72 ). The significance of the results was calculated by Student’s t-test or Anova and reported as P-value.
Definition of the pericentriolar region. We acquired images by confocal microscopy and drew a cube of 2 μm³ around the centrosome to define the pericentriolar region, as described in (73 (link)). The acquisition of z-stack imagines was performed collecting 6 slices for sample.