Monoclonal anti ha agarose

Cell extracts were prepared by adding CelLytic M reagent (Sigma-Aldrich) with 1% of Protease Inhibitor Cocktail Set III (Calbiochem) according to the manufacturer's protocols. Extracts were pre-cleared by incubation with 40 μl of rec-Protein A- or G-Sepharose 4B Conjugate (Invitrogen) and 2 μg of rabbit or mouse IgG (Santa Cruz) at 4°C for 1 h. For pulling down endogenous GRP78 or Flag-tagged GRP78 proteins, pre-cleared cell extracts were then incubated with 2 μg of anti-GRP78 (Proteintech) or anti-Flag M2 monoclonal antibody (Sigma-Aldrich) at 4°C for overnight followed by 40 μl of rec-Protein A- or G-Sepharose 4B Conjugate at 4°C for 2 h, respectively. For pulling down HA-GALNT6, pre-cleared cell extracts were incubated with 40 μl of monoclonal anti-HA-Agarose (Sigma-Aldrich) at 4°C for overnight. The beads were then spun down and washed 4 times with 1 ml of cell lysis buffer. Finally, immunoprecipitated proteins were released from the beads by boiling in sample buffer for 2 min or by adding elution buffer.

Yeast lysates were prepared by high-speed vortexing with glass beads in lysis buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5% NP-40, 4 mM β-glycerophosphate, 4 mM NaF, 4 mM Na₃VO₄, 4 mM Na₄P₂O₇, 2 mM DTT, and 1x protease inhibitor cocktail). Lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C. For immunoprecipitation, 10-µl antibody-coupled beads (monoclonal anti-HA agarose, Sigma-Aldrich) were mixed with lysate containing 2.5 mg protein that was diluted to 1 ml using lysis buffer without NP-40. After incubation at 4°C for 2 h, beads were pelleted and washed twice with lysis buffer and once with GEF buffer (20 mM Tris, 5 mM MgCl₂, 1 mM EDTA, 5 µM GDP, and 2 mM DTT). Beads were then incubated with 40 pmol GST-Cdc42 and 2.5 µCi GTP-γ-³⁵S in 45 µl GEF buffer for 15 min at RT, with gentle mixing every 3 min. Reactions were stopped with 0.5 ml ice-cold GEF buffer, after which beads were pelleted by centrifugation and supernatants were filtered through nitrocellulose filters (Protran BA 85, Whatman; GE Healthcare) to trap and assay protein-bound radioactivity. The beads containing immunoprecipitated protein were then incubated with SDS sample buffer (2% SDS, 2 mM β-mercaptoethanol, 4% glycerol, 40 mM Tris-HCl, pH 6.8, and 0.01% bromophenol blue) at 95°C, and Cdc24 levels were analyzed by immunoblot.

n vitro acetylation assays were performed as previously described [31 (link)] with modifications. Briefly, HA‐PCAF protein was immunoprecipitated from HEK293T cells using monoclonal anti‐HA‐agarose (A2095; Sigma Aldrich) after 48 h of transfection. The agarose was washed thrice in PBS containing 0.1% Tween 20 (PBST) buffer and once in acetylation buffer. Thereafter, the agarose was incubated with His‐Fascin in acetylation buffer with or without acetyl coenzyme A (CoA) at 30°C for 2 h. After the acetylation assays, the proteins were separated by SDS‐PAGE for Western blotting with acetylated lysine (anti‐Ac‐K, ICP0380; Immunechem, Columbia, Canada), HA (SC‐7392; Santa Cruz), and His antibodies (HT601‐01; Transgene, Beijing, China) using Odyssey Sa Infrared Imaging System (LI‐COR Biosciences). For acetylation site identification, the separated proteins were stained using Coomassie brilliant blue (24615; Thermo Fisher Scientific), and the protein bands were excised for MS analysis.