Sc 12894 r

The following antibodies were used in this work: guinea-pig anti-Duf extracellular (1:100) and rabbit anti-Duf extracellular (1:400; Weavers et al., 2009 (link)), rabbit anti-Pyd (1:100) and rat anti-PydEx5 (1:200; Carrasco-Rando et al., 2019 (link)), mouse anti-Fas3 [1:10; Developmental Studies Hybridoma Bank (DSHB), AB-528238], mouse anti-Peanut (1:50; DSHB, AB-528429) and mouse anti-Discs large (1:50; DSHB, AB-528203), rabbit anti-phospho-Histone H3 (Ser10) (1:100; Upstate/Sigma-Aldrich, 06-570), rabbit Cy3-anti-HRP (1:100; Jackson ImmunoResearch, AB_2340262), rabbit anti-GFP (1:300; Thermo Fisher Scientific, A-6455), rabbit anti-GFP (1:300; Chromotek, PABG1), rabbit anti-Zip (1:50; Kiehart and Feghali, 1986 (link)), rabbit anti-p-PKC (Thr410) (1:10; Santa Cruz Biotechnology, sc-12894-R), rabbit anti-PKCξ (C-20) (1:20; Santa Cruz Biotechnology, sc-216), rabbit anti-Patj (1:50; gift from Hugo Bellen, Bhat et al., 1999 (link)), rat anti-Sns (1:100; gift from Elizabeth Chen, Bour et al., 2000 (link)), anti-cubn2 (1:200; Atienza-Manuel et al., 2021 (link)) and anti-Sdk (1:200; Astigarraga et al., 2018 (link)). The following secondary antibodies were used: Alexa Fluor 488, 555 and 647 (Thermo Fisher Scientific) and DyLight 549 and 488 goat anti-rabbit IgGs (Vector Laboratories, DI-1549 and DI-1088). All secondary antibodies were used at 1:500 dilution.

Par3 (07-330, EMD Millipore), Par6 (ab49776 and ab6022, Abcam, Cambridge, MA, USA), aPKCζ (sc-17781, Santa Cruz Biotechnology, Dallas, TX, USA), p-aPKCζ–T560 (ab62372, Abcam, Cambridge, MA, USA), p-aPKCζ–T410 (sc-12894R, Santa Cruz Biotechnology, Dallas, TX, USA), actin (A2066, Sigma-Aldrich, St. Louis, MO, USA), F-actin BODIPY 558/568 Phalloidin (B3475, Invitrogen, Life Technologies Carlsbad, CA, USA), occludin (33-1500, Invitrogen, Life Technologies, Carlsbad, CA, USA), JAM-A S285 (sc-17430, Santa Cruz Biotechnology, Dallas, TX, USA), JAM-A Y280 (600-401-GN5, Rockland Immunochemicals Inc, Limerick, PA), JAM-A (361700, Invitrogen, Life Technologies, Carlsbad, CA, USA), and ZO-1 (61-7300, Invitrogen, Life Technologies, Carlsbad, CA, USA). Secondary antibodies used for immunofluorescence were Alexa Fluor (Life Technologies, Carlsbad, CA, USA). Dynasore (Sigma-Aldrich, St. Louis, MO, USA) was used at 80 µM. aPKCζ pseudosubstrate (539624, Millipore-Sigma, Burlington, MA, USA) was used at 5 and 10 µM.

The ACC‐LC‐176 and NCI‐H460‐LNM35 (LNM35) cell lines were previously reported.¹² Cancer cells were cultured in RPMI 1640 supplemented with 5 or 10% FBS (Gibco). The immortalized lung epithelial cell line BEAS‐2B was maintained as previously described.¹³ All were tested and confirmed to be free from mycoplasma contamination. An anti‐CERS6 antibody was purchased from Abnova (clone 5H7), anti‐RAC1 from Millipore (clone23A8), anti‐α‐tubulin from Sigma‐Aldrich, anti‐ceramide from Glycobiotech (S58‐9) and anti‐PKCζ and anti‐pPKCζ from Santa Cruz (SC216 and SC12894‐R, respectively). Anti‐mouse IgG and anti‐rabbit IgG were conjugated with HRP from Cell Signaling Technology. Anti‐mouse IgG and anti‐rabbit IgG conjugated with Alexa 488 or 568 were from Invitrogen. Fumonisin B₁ (Cayman Chemical) and myriocin (Sigma‐Aldrich) were also purchased. Sequence information of the oligonucleotide primers used for PCR and sequencing is provided in Table S1.