Anti ha antibody conjugated agarose beads

293T cells were lysed 48 hrs after transfection with CelLytic M cell lysis reagent (Sigma-Aldrich) containing a complete protease inhibitor cocktail (Roche Applied Science). For FLAG or HA tagged protein, whole-cell extract was incubated with anti-FLAG M2 antibody conjugated agarose beads (Sigma-Aldrich) or anti HA antibody conjugated agarose beads (Sigma-Aldrich) at 4°C overnight. Following three times washing with PBS, proteins bound to the beads were eluted by incubating with FLAG or HA peptide at 4°C for 1 h. For endogenous protein, whole-cell extract was incubated with a primary antibody at 4°C overnight. Protein A/G conjugated agarose beads were added to bind proteins / antibody complex followed by washing three times with PBS. Proteins bound to the beads were eluted by boiling in Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific).

Total cell protein was extracted with RIPA buffer containing 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA (pH 8.0), 150 mM NaCl, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, a 1:100 dilution of phosphatase inhibitor cocktail (Cell Signaling Technology, #5870 S), and a 1:100 dilution of protease inhibitor mixture (Bimake, #B14001). Cell lysates were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), blocked with 5% (w/v) nonfat milk, probed with the indicated primary antibodies and corresponding secondary antibodies, visualized using ECL Western blotting detection reagent (Millipore) and photographed using a Fuji Film LAS4000 mini-luminescent image analyzer. Anti-Flag antibody-conjugated agarose beads (Sigma-Aldrich, #A2220), Anti-Myc antibody-conjugated agarose beads (Sigma-Aldrich, #A7470), and anti-HA antibody-conjugated agarose beads (Sigma-Aldrich, #A2095) were used for the exogenous co-immunoprecipitation assay. Protein G Sepharose (GE HealthCare Company, #17-0618-01) was used for the endogenous co-immunoprecipitation assay. Image J software (National Institutes of Health) was used to quantify protein levels based on the band density obtained by Western blot analysis.

Anti-Flag antibody, anti-Myc antibody and anti-HA antibody-conjugated agarose beads were purchased from Sigma-Aldrich. The experimental procedures including Western blot and coimmunoprecipitation analysis have been described previously [79 (link)]. The Fuji Film LAS4000 mini luminescent image analyzer was used for photographing the blots. Multigauge V3.0 was used for quantifying the protein levels based on the band density obtained in the Western blot analysis.

Briefly, 6 × 10⁷ trophozoites were lysed in 1 mL of lysis buffer (1% Triton X-100, 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail, 100 μg mL⁻¹ TLCK, and 5 mM EDTA in TBS), centrifuged to remove unbroken cell debris before the addition of 20 μL of anti-HA antibody-conjugated agarose beads (Sigma-Aldrich, MA, USA), and then incubated on a rotator at 4°C overnight. The beads were recovered by centrifugation and washed three times with 1 mL lysis buffer. The precipitates were denatured in 1× SDS sample buffer for Western blotting or staining (55 (link), 57 (link)).

HEK 293T cells were transfected with pcDNA3-V5-PRKAA1 and pcDNA3-HA-FANCG or pcDNA3-HA-FANCA or the pcDNA3 empty vector using the Effectene Transfection Reagent (Qiagen, Valencia, CA, USA). The cells were treated 1 day later with 200 ng/mL MMC for 16 h and then lysed in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.3% Igepal CA-630, 0.2% Triton X-100, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors). Co-immunoprecipitation was performed as described previously [17 (link)]. Briefly, cell lysates were precleared with 10 μL protein A-agarose beads (Invitrogen, Carlsbad, CA, USA) and incubated with anti-HA antibody-conjugated agarose beads (Sigma, St. Louis, MO, USA), anti-V5 antibody-conjugated agarose beads (Sigma), or an anti-FANCA antibody (A301-980A, Bethyl Laboratories, Montgomery, TX, USA) with protein A-agarose beads for 18 h at 4°C. The beads were washed with lysis buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Co-immunoprecipitated proteins were detected by immunoblotting with anti-V5 (Invitrogen), anti-FANCA (Bethyl Laboratories), anti-FANCG (Novus Biologicals, Littleton, CO, USA), or anti-AMPKα (Cell Signaling Technology, Danvers, MA, USA) antibodies.