Mouse anti lbpa

Filter-grown cells were fixed in 2% paraformaldehyde for 10 min, blocked in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), and incubated with specific primary antibodies for 1 h: mouse monoclonal anti–acetylated tubulin clone 6-11B-1 (1:1000; Sigma-Aldrich), rat monoclonal anti-tyrosinated α-tubulin (1:200; Santa Cruz), mouse anti-LBPA (1:500; Echelon Biosciences, Salt Lake, UT; Z-LBPA), mouse anti-ceramide (1:10; Enzo Life Sciences, Farmingdale, NY) and rat anti–Z0-1 (1:3000; Xu et al., 2012 (link)). Alexa Fluor secondary antibodies were used at 1:500 and rhodamine–phalloidin (Cytoskeleton, Denver, CO; PHDR1) at 1:200. Filters were mounted under coverslips on glass slides under Vectashield (Vector Labs, Peterborough, UK), sealed and visualized with an Andor (Belfast, UK) Revolution XD spinning-disk confocal microscope using a 60×/1.4 NA oil objective with identical exposures and gains for each antibody. Acetylated tubulin-labeled stable microtubules (≥5 μm in length) after nocodazole treatment were analyzed manually in the Surpass mode of Imaris (Bitplane, South Windsor, CT).

AP staining was performed using an ES-AP detection kit (EMD Millipore) according to manufacturer’s recommendations. SA-β-gal activity was detected using SA-β-gal staining kit (Cell Signaling Technology) according to manufacturer’s protocol. For immunocytochemical analysis, we used anti-SSEA4, TRA-1-60, TRA-1-81 (mouse, 1:100; EMD Millipore), anti–β-III-tubulin (mouse, 1:400; EMD Millipore), rabbit anti–LC-3B (1:200; Cell Signaling Technology), rabbit anti-ASM (1:1,000, Abcam), mouse anti-LAMP1 (1:100; Abcam), and mouse anti-LBPA (1:500; Echelon).