Biotin bmcc

Protein palmitoylation detection was performed by immunoprecipitation and acyl‐biotin exchange as described previously.^[81] Briefly, cells were lysed in lysis buffer (LB) (1% NP‐40, 50 mM Tris pH 7.5, 150 mM NaCl, 10% Glycerol) supplemented with protease inhibitor, PMSF and 50 mM N‐ethylmaleimide (NEM) (Acros Organics, 156100100). Equal amounts of lysates were incubated with the STING antibody overnight at 4 °C followed by 1 h incubation with 60 µL Protein A/G XPure Agarose Resin, or incubated with Flag agarose beads for 3–4 h at 4 °C. Beads were resuspended in LB+10 mM NEM and split into triplicates. 1/3 of beads were used as ‐HAM control and 2/3 were used as +HAM treatment. Beads were resuspended with LB (pH 7.2)+0.1% SDS quickly, washed with LB (pH = 7.2) three times, resuspended in LB (pH 7.2) with or without 1 m hydroxylamine (HAM) (Thermo Scientific, A15398.30) and incubated at room temperature for 1 h. Then, beads were washed with LB (pH 6.2) once and resuspended in LB (pH = 6.2)+2 µM Biotin‐BMCC (Thermo Scientific, 21900) at 4 °C for 1 h. Beads were then washed with LB (pH = 6.2) once and LB (pH 7.5) for three times. Beads were boiled and resolved by SDS‐PAGE and immunoblotted with streptavidin‐HRP antibody to detect biotin‐labeled palmitoylated STING.

This method was adapted from a previous publication [22 (link)]. Briefly, CD44 protein was immunoprecipitated from lysates via overnight incubation at 4°C with 3 μg mouse anti-human CD44 antibody as described elsewhere [18 (link)]. Antibody–protein complexes were collected by 3-hour rotation at 4°C with 50 μg protein G-sepharose (Sigma-Aldrich), in the presence of 50 mM N-ethylmaleimide (Sigma-Aldrich), to covalently block sulfhydryl groups. Complexes were subsequently divided into two fractions, for treatment with and without 1 M hydroxylamine (Sigma-Aldrich) at room temperature for 1 hour (pH 7.40) to cleave free palmitate groups. Samples were then incubated with 1 μM EZ-link biotin-1-biotinamido-4-(4′-(maleimidomethyl)cyclohexanecarboxamido)butane (biotin-BMCC; ThermoScientific) at pH 6.2 for 1 hour at room temperature, to label the reactive cysteine residues. Labelled CD44 was released from the beads via incubation (100°C/5 minutes) in 2× reducing Lamelli sample buffer, and was subjected to SDS-PAGE and immunoblotting. biotin-BMCC-labelled CD44 was detected with streptavidin-HRP, followed by antibody, to visualise total CD44.

Palmitoylation assay was performed using the acyl-biotin exchange method as described (37 (link)). In brief, HEK293T cells were transfected with myc-tagged WT or CA mutant of PLSCR2 for 48 h. To block reactive cysteines, cells were lysed in N-ethylmaleimide 50 mM (NEM, Sigma) containing cold IP lysis buffer. Following centrifugation at 12,000 g for 10 m, the supernatants were incubated with anti-myc antibody conjugated protein-G beads (Millipore) overnight at 4°C. The beads were rapidly washed in IP lysis buffer (pH 7.4) containing 10 mM NEM and then treated with or without 1 M hydroxylamine (HAM) for 1 h at room temperature to cleave palmitate from cysteines. After washing three times with pH-adjusted IP lysis buffer (pH 6.2), the beads were exposed to sulfydryl-specific biotinylating reagent containing 1 µM biotin-BMCC (Thermo Fisher Scientific) in IP lysis buffer (pH 6.2) for 2 h at 4°C. The biotinylated proteins were eluted and analyzed by western blotting using streptavidin-conjugated HRP and ECL.

Protein palmitoylation was determined by the ABE method (45 (link)). In brief, cells were lysed with cold RIPA lysis buffer containing 50 mM N-ethylmaleimide (NEM). Samples were immunoprecipitated by adding CD36 antibody overnight and Protein-A/G Sepharose beads for 3 h at 4 °C in a rotating shaker. The precipitate was blocked in lysis buffer with 10 mM NEM followed by washing to remove unbound NEM with stringent buffer (lysis buffer, 10 mM NEM, and 0.1% SDS) and lysis buffer. The sample was split into two equivalent samples: one was incubated with 1 M hydroxylamine (HAM) treatment (+HAM sample) for 50 min at room temperature to cleave thioester bonds at palmitoylated cysteines; another sample without HAM was used as a control (−HAM sample). After one wash in lysis buffer (pH 6.2), the beads were incubated with 500 μl lysis buffer containing 1 μM biotin-BMCC (Thermo Fisher Scientific) with rotation to selectively label the palmitoylated cysteines. The resulting thiol-biotinylated proteins were further detected with streptavidin–horseradish peroxidase (Beyotime) by Western blotting.