Protein g agarose beads

HEK293T cells cultured in 6-well plates were transfected with the respective plasmids (3 μg/each) by using Lipofectamine LTX and Plus reagents (Invitrogen) according to the manufacturer's instructions. At 48 h posttransfection, cells were washed three times with cold phosphate-buffered saline (PBS) (pH 7.4) and lysed with IP buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol [Pierce, Rockford, IL]) containing a complete protease inhibitor cocktail (1 tablet/50 ml) (Roche Diagnostics GmbH, Mannheim, Germany) for 30 min on ice. The supernatants were then mixed and rocked at 4°C overnight with the indicated primary antibodies. Protein G-agarose beads (Roche) were added, and the mixture was rocked for 6 to 8 h at 4°C. The IgG-agarose beads were washed four times with 1 ml of wash buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40), and immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blotting.

HeLa cells or mouse livers were lysed, centrifuged, and pre-cleared with protein G agarose beads (Roche). Supernatants were collected and 1 μg of antibody was added. After overnight incubation, 30 μl of 50% slurry of protein-G agarose beads were added, and the samples incubated at 4 °C for 2 hr. The agarose beads were pelleted by centrifugation, washed three times with ice-cold washing buffer (20 mM HEPES [pH 7.4], 0.1% NP-40, 150 mM NaCl, 0.25% sodium deoxycholic acid, 1 mM EDTA, 1 mM PMSF, 1 μM TSA, 10 mM NAM, and protease inhibitor cocktail [Roche]), resuspended in electrophoresis sample buffer (0.09 M Tris-Cl [pH 6.8], 20% glycerol, 2% SDS, 0.1 M DTT, and 0.02% bromophenol blue), boiled for 5 min, and subjected to electrophoresis, and immunoblotting.

ChIP was conducted as previously reported [19 (link)]. Briefly, cells were cross-linked (using 1% formaldehyde) and lysed (in ChIP lysis buffer: HEPES-KCl 50 mM, pH 7.5; NaCl 140mM; EDTA 1mM; Triton X-100 1%; sodium deoxycholate 0.1% and SDS 0.1%). They were then sonicated for the shearing of chromatin into smaller (200-500-bp) fragments. The generated fragments were then immunoprecipitated with either control rabbit IgG antibody (Cell Signaling) or ChIP-grade antibody (anti-STAT3; Millipore), and incubated with protein G agarose beads (Roche Applied Science). This was followed by reversal of cross-linking by Proteinase K treatment, and recovery of DNA. SYBR green dye (QIAGEN, Valencia, CA) was used to quantitate PCR products. Primers used for MMP-9 gene promoter were: 5’-tggggaggatatctgacctg-3’/5’-ttgcaaactgcagagcttgt-3’.

For Ctr-infected cells, Bak protein was immunoprecipitated according to the protocols described in [62 (link)]. For IP non-activated Bak, anti-Bak(7D10) (a kind gift from Dr. David Huang, WEHI) was used, and cells were harvested and directly lysed in buffer containing 1% CHAPS (Carl Roth, #1479.2). For IP of activated Bak, cells were treated as indicated the presence of caspase inhibitor QVD-OPh (Gentaur (ApexBio), #GEN2269261) (10 µM). Cells were then harvested and subsequently lysed in buffer containing 1% CHAPS as reported [62 (link)]. Following centrifugation at 13,000xg, protein concentration of the supernatants was quantified using Bradford assay (Bio-Rad) and equal amounts of protein were precleared with protein G agarose beads (Roche, #11719416001) for 60 min at 4 °C with constant agitation. Precleared lysates were incubated with Bak antibodies (either Bak(aa23-38) or Bak(Ab-1)) or with Bak(7D10) for 120 min at 4 °C, followed by incubating with protein G agarose beads (Millipore Sigma, #11719416001) for an additional 60 min under constant agitation to allow the antibodies to bind to the beads and samples were collected and separated by SDS-PAGE and immunoblotted.

Cells were lysed in 30 mM triethanolamine, 1% NP-40, 50 mM NaF, 2 mM NaV₂O₅, 1 mM, Na-tetrathionate, 5 mM EDTA, 5 mM EGTA, 100 mM N-ethylmaleimide, 50 mM NaCl, 10 uM Pepstatin A, Roche Protease inhibitor tablet (pH 7.4) for 30 min at 4°C. Lysates were centrifuged in a microcentrifuge for 20 min at 12,000 rpm at 4°C to remove cellular debris. Protein was measured by a Bradford assay, and samples were either flash-frozen in SDS sample buffer or processed for immunoprecipitation.
Abl2 was immunoprecipitated from lysates with goat anti-Abl2 antibodies (Santa Cruz C20) and Protein-G Agarose Beads (Roche), following the manufacturer’s instructions. Protein was eluted from the beads with SDS sample buffer and fractionated by SDS-PAGE. Proteins were transferred onto PVDF membranes, which were probed with antibodies to Abl2 (Rabbit polyclonal, Proteintech). We used antibodies to pan-Cadherin (Cell Signaling Technologies) and Myosin heavy chain My32 (Sigma) to ensure equal loading of proteins in whole cell lysates.

Cells were lysed in NP40 lysis buffer and then cell lysates were centrifuged at 12,000 g, 4 °C for 20 min. 20 µL protein G agarose beads (Roche, 11243233001) were added to the supernatants for preclear. The supernatants were incubated with protein G agarose beads and indicated antibodies at 4 °C for 8 h. Then immunocomplexes were centrifuged at 4 °C for 3 min and NP-40 lysis buffer were used to wash the precipitates three times before subjected to western blot assay.
For western blot assay, proteins were separated on 10% or 12% SDS-PAGE gel where appropriate. Proteins were then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the indicated antibodies and washed 3 times with TBST (0.05% Tween-20, 150 mM NaCl and 20 mM Tris-HCl). Lastly, PVDF membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, 31460) or horseradish peroxidase-conjugated anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).