Ezview red anti flag m2 affinity gel

Lysates containing equal amounts of protein as determined by the Bradford assay or bicinchoninic acid assay (BCA assay) were combined with EZview Red anti-FLAG M2 affinity gel (Sigma, F2426) as per the manufacturer’s instructions. For V5-AMPKγ1 immunoprecipitation, protein A/G agarose (Santa Cruz, sc-2003) was combined with monoclonal anti-V5 antibody (Sigma, V8012), which was used at 1ug of antibody/1 mg of protein, and rotated overnight at 4°C. The supernatant was removed and the affinity gel or agarose pellets were washed by adding 1mL of 0.1% NP40 lysis buffer followed by 5 minutes rotation at 4°C for a total of 4 times. For immunoprecipitation using the membrane fraction, affinity gel pellets were washed with 1mL of 0.1% NP40 lysis buffer, followed by 5 minutes rotation at 4°C for 3 times and with 1mL 0.5% NP40 lysis buffer followed by 5 minutes rotation at 4°C for one time. Detergent was then removed from the membrane fraction using Pierce Detergent spin columns. FLAG-K1 and/or FLAG-K1 domain deleted mutants were eluted using 3X FLAG peptide (Sigma, F4799) as per the manufacturer’s instructions. Laemmli buffer (2X) was added to the FLAG-K1eluate or directly to the V5-AMPKγ1/agarose samples (1:1) and all samples were heated at 100°C for 6 minutes. Proteins were resolved by SDS-PAGE followed by Western blot.

HeLa cells were homogenized in a NP-40 lysis buffer containing 25 mM Hepes, pH 7.4, 10% glycerol, 50 mM sodium fluoride, 10 mM sodium phosphate, 137 mM sodium chloride, 1 µM sodium orthovanadate, Protease Inhibitor Cocktail (EMD CHEMICALS INC. Rockland, MA, USA) and rocked for 10 min at 4°C. Homogenates were centrifuged for 10 min at 13,000×g at 4°C, and supernatants were collected. Protein concentration was determined using the BCA method. Lysates from HeLa cells expressing Flag-LKB1-WT were incubated with 50 µl of EZview Red ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) for O/N (18 h). The same amount of lysates from HeLa cells expressing Fyn-WT, W119A or R176K-V5 was added for 2 h. Samples were separated onto 10%SDS-PAGE followed by immunoblotting using anti-Flag and anti-V5 antibodies.
C2C12 myotubes were incubated with either 11R-random or 11R-LKB1-WT peptide for 30 min and homogenized. LKB1 was immunoprecipitated using the LKB1 monoclonal antibody and phosphorylation levels of tyrosine 261 and 365 of LKB1 were assessed by immunoblotting, using specific antibodies.

The indicated FIP200 and RIG-I domains were cloned into pGEX-5X-3 (GE Healthcare, # 28-9545-55) to fuse with a GST tag, pET28b(+) (Novagen, # 69865-3) to fuse with a His tag, pT7-FLAG-2 (Sigma, # P1243) for a FLAG tag, or pMXB10 (New England Biolabs, # E6901S) for a MBP tag. These constructs were transfected into BL21 (DE3) E. coli (New England Biolabs, # C2527I) and cultured in LB broth at 20 °C. IPTG (0.4 mM) was added to induce protein expression. MBP-tagged proteins were purified using the IMPACT kit (New England Biolabs, # E6901S), and the MBP pull-down assays were performed using the anti-MBP Magnetic Beads New England Biolabs, # E8037S). FLAG-tagged proteins were purified by using the EZview Red Anti-FLAG M2 Affinity Gel (Sigma, # F2426). The GST Protein Interaction Pull-Down Kit (ThermoFisher Scientific, # PI21516) was used for GST-tagged protein purification and GST pull-down assays. The His-Spin Protein Miniprep kit (Zymo Research, # P2002) was used for His-tagged protein purification and His pull-down assays.

Larval lysates were prepared by crushing animals in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40) with 1× protease inhibitor cocktail (Invitrogen) and clearing the lysate by centrifugation at 13,000 RPM for 10 min at 4°C. S2 cell lysates were prepared by suspending cells in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40) with 10% glycerol and 1× protease inhibitor cocktail (Invitrogen) and disrupting cell membranes by pulling the suspension through a 25 gauge needle (Becton Dickinson). The lysate was then cleared by centrifugation at 13,000 RPM for 5 min at 4°C. Western blotting on lysates was performed using standard protocols. Rabbit anti-dSMN serum [28] (link) was affinity purified. For Western blotting, dilutions of 1 in 2,500 for the affinity purified anti-dSMN, 1 in 10,000 for anti-α tubulin (Sigma), 1 in 10,000 for monoclonal anti-FLAG (Sigma), 1 in 10,000 for polyclonal anti-Myc and 1 in 5000 for monoclonal anti-Myc (Santa Cruz) were used. Anti-FLAG antibody crosslinked to agarose beads (EZview Red Anti-FLAG M2 affinity gel, Sigma) or anti-Myc antibody crosslinked to agarose beads (Sigma) were used to immunoprecipitate FLAG and Myc tagged proteins from cells.