Protein a g plus

Non-denaturing immunoprecipitation was performed by bringing about the lysis of cells in 1 ml of radio immune precipitation assay buffer (RIPA) for 30 min at 4 °C, scraped followed with centrifugation at 16,000g for 20 min at 4 °C. The supernatant was rotated with anti-Nrf2 antibody overnight at 4 °C on rocker. Protein A/G PLUS (Santa Cruz Biotechnology) was added (30 μl), and samples were incubated for 2 h at 4 °C. Immunoprecipitates thereafter were pelleted, washed three times with 1 ml of radioimmune precipitation assay (RIPA) buffer, boiled in Laemmli buffer and were analyzed by western blotting with anti-ubiquitin antibody [7] (link).

Western blot was performed according to the previously published protocol^{29 (link)}. For coimmunoprecipitation cells were lysed in hypotonic buffer, followed by nuclear lysis. Protein lysates were incubated in pulldown antibody for 2 h at 4 °C and beads (Protein A/G PLUS, Santa Cruz; normal mouse antibody (CST) and beads without antibody served as control) added for one more hour. Beads were washed with nuclear lysis buffer and SDS-PAGE performed. Buffer recipes are listed in the Supplementary information. ChemiDoc™ Touch Imaging (Bio-Rad) System was used for detection. Quantification of protein amount was done by Image Lab^TM Software (Bio-Rad).

Cells derived from our experimental conditions were lysed in radioimmunoprecipitation assay (RIPA) buffer and 30 μg of total proteins were loaded on precast SDS-PAGE gels (BioRad), separated and transferred onto PVDF membrane. Immunoblots were incubated overnight in 5% BSA or milk in T-PBS at 4°C and probed with primary and appropriate secondary Horseradish Peroxidase (HRP)-antibodies. Immunoprecipitation was performed on 500 mg of total proteins in 500 ml of RIPA buffer using 20 μl of protein A/G plus (Santa Cruz Biotechnology) per samples, following manufacturer's instruction.
Detection of PKM2 redox state, N-(biotinoyl)-N’-(iodoacetyl) ethylene diamine BIAM labeling was performed as previously described [34 (link)]. Briefly, cells were rapidly rinsed in liquid nitrogen and exposed to RIPA buffer containing 100 μM of BIAM diamine and incubated for 15 min at room temperature. Lysates were then clarified by centrifugation and 500 μg of each sample were suspended into 500 μl of RIPA buffer and immunoprecipitated using 20 μl of Streptavidin-agarose beads (Sigma). Immunocomplexes labelled with BIAM were separated by SDS–PAGE and the biotinylated/reduced fraction was revealed with HRP-conjugated.

HEK293T cells cotransfected with the indicated plasmids were lysed using lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 mM Tris pH 8, 1 mM sodium orthovanadate) supplemented with protease (Sigma) and phosphatase inhibitor cocktails (Roche PhosSTOP easy pack). The cells were incubated with lysis buffer on ice. The lysates were then clarified by centrifugation (15 min, 18,800 g, 4˚C). Protein lysates were precleared with protein A\G plus (Santa Cruz Biotechnology). Rabbit anti-V5 antibodies (1:200) or M2 affinity gel beads (Sigma) were added for overnight incubation. The next day A\G beads were added for 1.5 h at 4˚C. The samples were then washed three times using lysis buffer. The samples were eluted by 5 min boiling in sample buffer and analyzed by western blot.

Cortical neurons and brain cortex from siah2+/+ control mice and mice exposed to tMCAO were homogenized in lysis buffer containing 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1% Triton X-100, 100 mM NaF, 100 mM Na₃VO₄, 5 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 μg/ml pepstatin. One milligram of lysate was precleared using protein A/Gplus (Santa Cruz, Dallas, TX) for 1 h at 4 °C with constant rotation and centrifuged for 2 min at 8000 rpm. The cell lysates (1 mg) were cleared by centrifugation at 15,000 x g for 15 min and were immunoprecipitated with anti-Siah2 mouse antibody (Sigma-Aldrich, 1:100). An aliquot of cell lysate (100 μg) or immunoprecipitates were resolved by SDS-PAGE gel and transferred onto nitrocellulose membrane. Immunoblot analysis was performed using anti NCX3 and VDAC antibodies, as previously described [1 (link)]. Chemio-luminescent (ECL) signals were quantified by Chemi Doc Imaging System (Biorad).
Regarding lysates from brain tissue, 1 mg of precleared lysate was immunoprecipitated with an anti-Siah2 mouse antibody (Sigma-Aldrich, 1:100) using the same experimental procedure described above. Finally, total lysates or immunoprecipitates were resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane. Immunoblot analysis was performed using anti-NCX3 and anti-VDAC antibodies.

The transfected cells were lysed in a lysis buffer containing 5 mM EDTA, 250 mM NaCl, 50 mM HEPES, and 0.1% NP40. The lysates were subjected to SDS-PAGE, transferred to PVDF membrane, and detected by Western blot using anti-HERG antibodies (F-12 from Santa Cruz, Delaware Avenue, CA, USA and APC016 from Allomone labs, Jerusalem, Israel). Co-immunoprecipitation experiments were performed using anti-GFP antibody (Invitrogen, Carlsbad, CA, USA) and protein A/G plus (Santa Cruz, Delaware Avenue, CA, USA). The immunoprecipitated proteins were released from the beads and analyzed by sequential Western blot analysis.