Mouse monoclonal antibody against flag

Equivalent amounts of total cellular lysates were separated on 4% to 12% Tris-Glycine gels (Invitrogen) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% BSA in TBS-T (Tris-buffered saline-0.1% Tween 20) for 1h at room temperature (RT), and then followed by incubation with primary antibodies overnight at 4°C. A rabbit monoclonal antibody against LRRK2 (Abcam) was used at 1:1,000 dilution. A mouse anti-GAPDH monoclonal antibody (Thermo Fisher) was used at a 1:10,000 dilution. A mouse monoclonal antibody against FLAG (Sigma) was used at 1: 2,500. A rabbit anti-phospho-RIP2 (S176) (abm) was used at 1:1,000. After incubation, membranes were washed 5 times for 5min with TBS-T and were further incubated with appropriate secondary antibodies coupled to horseradish peroxidase for 1h RT. Upon extensive washing, the membrane was developed with enhanced chemiluminescence detection reagents (Bio-Rad), followed by imaging using a ChemiDoc Touch imaging system (Bio-Rad).

Immunogold TEM was carried out as previously described.^{47 (link)} In brief, HeLa cells were transfected with BECN1-flag for 24 h, and then fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min. Cells were washed in PBS buffer three times, incubated in 0.1 M Na-PB containing 0.25% saponin and 5% BSA for 30 min, and then incubated in PB containing 0.005% saponin, 10% BSA and 0.1% cold water fish skin gelatin. Cells were treated with mouse monoclonal antibody against flag (Sigma-Aldrich) in the blocking solution overnight. Cells were then washed in PB containing 0.005% saponin for 5 min three times and incubated with anti-mouse IgG that was conjugated to colloidal gold (10 nm diameter) in the blocking solution for 2 h. Cells were washed with PB for 5 min three times and fixed with 1% glutaraldehyde in PB for 10 min. After washing in distilled water, cells were postfixed in 0.5% OsO₄ for 2 h at 4 °C, washed in distilled water, incubated with 50% ethanol for 10 min and stained with 2% uranyl acetate in 70% ethanol for 2 h. Cells were further dehydrated with a graded series of ethanol and were embedded in epoxy resin. Ultrasections were doubly stained with uranyl acetate and lead citrate.

Mouse monoclonal antibody against Flag and anti-Flag M2 affinity gel were purchased from Sigma (St. Louis, MO). Rabbit monoclonal antibody against HA Tag and phospho-Smad2 (Ser465/467) were obtained from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibody against PGM1 and rabbit polyclonal antibody against FOXJ2 were purchased from Abcam (Cambridge, MA). Flag peptide and mouse monoclonal antibody against Actin were obtained from Abclonal Technology (Wuhan, China). Rabbit polyclonal antibody against CNBP and rabbit polyclonal antibody against GRP78 were obtained from Proteintech Group (Wuhan, China). Mouse monoclonal antibody against p53 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). D-Galactose and Tunicamycin were obtained from MedChemExpress (Monmouth Junction, NJ). TGFβ recombinant protein was bought from Peprotech (New Jersey). Puromycin and hygromycin were bought from EMD Biosciences (SanDiego, CA). In vitro DNA transfection reagent was purchased from Signagen Laboratories (Rockville, MD). GelCode Blue Stain Reagent was obtained from Pierce (Rockford, IL).

Mouse monoclonal antibody against Flag, anti-Flag antibody conjugated agarose beads, dithiothreitol (DTT), iodoacetamide (IAA) were from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal antibody against β-actin was from Santa Cruz (Santa Cruz, CA, USA). Mouse monoclonal antibody against PKCζ and rabbit polyclonal antibody against PPP2CA were from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine 2000, BCA reagents, and Protein G agarose beads were purchased from Invitrogen. Enhanced chemiluminescence reagents were from Pierce Biotechnology. Protease Inhibitor Cocktail tablets were from Roche Diagnostics (Indianapolis, IN, USA). Sequencing grade modified trypsin was purchased from Promega (Madison, WI, USA). LC-MS grade water and acetonitrile were bought from Merck (White-house Station, NJ, USA).

The p3xFlag-GV141-P/CAF plasmid was purchased from Shanghai GeneChem Co., Ltd. The fragment of BRCA2-290-453aa-wildtype was cloned from pDEST26 -BRCA2 gifted from Dr. Dongyi Xu (Peking University). Using BM seamless cloning kit (Biomed, China), the truncation was cloned into pXJ40-HA. The construct with a mutation in BRCA2-290-453aa-N372H was generated as described previously (Yang et al., 2015 (link)).
Anti-Flag M2 agarose affinity gel and mouse monoclonal antibody against Flag was purchased from Sigma (St. Louis, MO, United States). Antibody against HA and antibody against 53BP1 were from Abcam.

Over night bacterial culture was diluted 1:100 in LB or 1:30 in LB supplemented with 5 mM EGTA, and cultured for 3.5 h. The supernatant and pellet were separated by centrifugation. Samples from equal numbers of bacteria were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and probed with a rabbit polyclonal antibody against ExoS or a mouse monoclonal antibody against FLAG (Sigma). Signals were detected with the ECL-plus kit (Millipore).