Mouse anti acetylated tubulin 6 11b 1

Antibodies were obtained from the indicated suppliers: goat anti-Rootletin for immunostaining (sc-67824) and mouse anti-Rootletin for immunoblotting (sc-374056; Santa Cruz Biotechnology, CA); mouse anti-acetylated tubulin (6–11B-1), mouse anti-FLAG (M2), and mouse anti-gamma-tubulin (T3559; Sigma-Aldrich, St. Louis, MO); mouse anti-Myc (9B11; Cell Signaling Technology, Danvers, MA); and rabbit anti-Pericentrin 2 (ab4448) and HRP-conjugated anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab9385; Abcam, MA). Species-specific secondary antibodies were conjugated to Alexa Fluor 488 or 555 (Life Technologies, Grand Island, NY). Western blot analyses were performed as described previously31 (link).

Dense monolayers were grown in 12-well chamber slides (removable) (#81201, Ibidi, Martinsried, Germany), fixed for 20 min in 4% paraformaldehyde in PBS, permeabilized for 15 min in 0.25% Triton-X-100 in phosphate-buffered saline (PBS), blocked with 5% normal goat serum (Merck Millipore, Darmstadt, Germany) in PBS and stained cell–cell junctions, polarity or markers of origin, F-actin and nuclei, and mounted with coverslips using Shandon Immu-Mount (Thermo Fisher Scientific, Waltam, MA, USA). Antisera were rabbit anti-E-cadherin (#3195, 24E10; Cell Signaling, Frankfurt, Germany), mouse anti-zonula occludens-1 (#339100, ZO1-1A12; Thermo Fisher Scientific), rabbit anti-pericentrin (#ab4448; Abcam, Cambridge, United Kingdom), mouse anti-acetylated tubulin (6-11B-1; Sigma-Aldrich), mouse anti-aquaporin 1(#NB600-749, 1/A5F6; Novus Bio, Abingdon, United Kingdom), rabbit anti-aquaporin 2 (TA335241; OriGene, Herford, Germany), Alexa Fluor 488 donkey anti-mouse lgG (H + L) (A-21202), Alexa Fluor 555 goat anti-mouse lgG (H + L) (A-21422) and Alexa Fluor 555 goat anti-rabbit lgG (H + L) (A-21428; Life Technologies, Carlsbad, CA, USA). Alexa Fluor 660 Phalloidin, (A-22285; Life Technologies) was used for F-actin and DAPI (0.25 µg/ml; Sigma-Aldrich) to stain nuclei.