Article Title: Palmitoylation controls trafficking of the intracellular Ca2+ channel MCOLN3/TRPML3 to regulate autophagy
Figure Lengend Snippet: Palmitoylation sites and PATs of MCOLN3/TRPML3. (a) Schematic of MCOLN3/TRPML3 showing palmitoylation sites (gray and red circles) predicted by CSS-Palm v2.0 and sequence alignments of the indicated cysteine residues across different species and among MCOLN/TRPML subfamily members. (b) MCOLN3/TRPML3 palmitoylation assay. HEK293T cells expressing GFP-tagged MCOLN3/TRPML3 (upper panel) or HEK293T and SW13 cells (lower panel) were either treated with DMSO (control) or metabolically labeled with 100 μM 17-ODYA. After 6 h, membrane fractions were prepared followed by reaction between biotin-azide and 17-ODYA using click chemistry. Biotinylated proteins were affinity isolated using streptavidin beads and eluates were analyzed by western blot using anti-GFP antibody or anti-MCOLN3/TRPML3 antibody to detect palmitoylated proteins. Tris or HN were added to the reaction mixture and incubated for 1 h at RT before adding the beads. Input samples were 1% of protein lysate used in the click chemistry reactions. (c) MCOLN3/TRPML3 and cysteine mutant palmitoylation assays. HEK293T cells expressing GFP-tagged MCOLN3/TRPML3 and the indicated single or triple cysteine mutants were assayed as in (b). (d) Quantified data of ratios of palmitoylated MCOLN3/TRPML3s to total MCOLN3/TRPML3 protein levels (n = 3, * p
Article Snippet: The following antibodies were used: anti-LAMP1 (BD biosciences, 555798), anti-GFP (Invitrogen, ), anti-LC3 (Thermo Fisher Scientific, PA1-16930), and anti-MCOLN3/TRPML3 (Alomone Labs, ACC-083).
Techniques: Sequencing, Expressing, Metabolic Labelling, Labeling, Isolation, Western Blot, Incubation, Mutagenesis