Pureproteome protein g magnetic beads

In brief, EPCs were washed three times with PBS and total proteins were harvested by the NP-40 immunoprecipitation (IP) cell lysis buffer (Boster, China, AR0107). The protein complex was collected with PureProteome protein G magnetic beads (Millipore, MA, LSKMAGG10) according to the manufacturer’s protocol. Proteins were incubated with anti-Bad antibody, anti-Bcl-xL antibody, or control normal rabbit IgG at 4 °C overnight with a dilution ratio of 1:50 respectively. After washed for three times, the beads were added to SDS-loading buffer and boiled for 5 min. The samples were then loaded to SDS-PAGE gel, and the protein level of Bcl-xL and Bad was detected by western blot as described below.

Cells were lysed in RIPA buffer with a protease inhibitor cocktail, and cell extracts (1 mg) were immunoprecipitated with anti-FAK or anti-FLAG M2 antibodies (1 μg) for 24 h. The corresponding rabbit and mouse IgGs (Millipore) were used as the control antibodies. The bound samples were precipitated with PureProteome Protein G Magnetic Beads (Millipore) for 1 h at 4 °C, and the immunoprecipitated products were collected for Western blot analysis.

SH-SY5Y cells incubated for 24 h with either vehicle or VPA were treated with either vehicle or proNGF for 2 h. The cells were lysed with ice-cold RIPA buffer supplemented with 1% Triton X 100. Following centrifugation at 10,000 x g for 10 min at 4 °C, the supernatant (~ 500 µg of protein) was incubated overnight at 4 °C with either anti-p75NTR rabbit antibody (1:75) (Cell Signaling Technology, Danvers, MA, USA) or preimmune rabbit IgG (Santa Cruz Biotechnology). Thereafter, 50 µl of Pure Proteome Protein G magnetic beads (Millipore, Burlington, MA, USA) were added and samples incubated at 4 °C for 3 h with continuous rotation at 4 °C. The beads were washed 5 times with ice-cold PBS/0.1% Tween 20 buffer. After the last wash the pellet was resuspended in 2 x sample buffer and boiled for 5 min.

Whole-cell lysates were collected by adding SDS to the sample buffer. After extensive sonication, the samples were boiled for 10 min and subjected to SDS-PAGE. The proteins were then transferred to nitrocellulose membranes and analyzed using immunoblotting (ECL Plus, GE Healthcare, Barcelona, Spain). For immunoprecipitation assays, the cells (~5 × 10⁶) were lysed in 500 μl of lysis buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.4% NP40, 10 mM MgCl₂) supplemented with protease and phosphatase inhibitor cocktails (Sigma). The cell extracts were cleared by centrifugation (20 000 × g for 15 min) and then diluted with 500 μl of dilution buffer (50 mM Tris, pH 8.0, 0.4% NP40, 2.5 mM CaCl₂) supplemented with protease and phosphatase inhibitor cocktails and DNase I (Sigma). The extracts were pre-cleared in 30-min incubations with 20 μl of Pure Proteome Protein G Magnetic Beads (Millipore) at 4 °C while being rotated. The antibodies (as indicated in the figure legends) were then added to the pre-cleared extracts. After incubation for 1 h at 4 ºC, 50 μl of Pure Proteome Protein G Magnetic Beads were added, and the extracts were further incubated for 20 min at 4 °C with rotation. After extensive washing, the bound proteins were analyzed using western blots. The unbound extracts were used as the positive inputs to determine protein loading.

U2OS cells were seeded into 15cm plates at a density of 2×10⁶ cells/plate and treated with 10Gy of γ radiation for 16 h or 72 h. ChIP assays were performed as described before [41 (link)]. After sonication, 10% of each sample was saved as total input, 45% of each sample was incubated with 5 μg of anti-p53 antibody (Cat# sc-126, Santa Cruz, TX), and the remaining 45% of each sample was incubated with 5 μg of normal mouse IgG (Cat# sc-2025, Santa Cruz, TX) at 4°C overnight and then with 50 μl (bead volume) of Pure Proteome proteinG magnetic beads (Cat# LSKMAGG02, Millipore, CA) at 4°C for 4 h. After wash and reverse crosslinking, DNA was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in H₂O. 2μl of ChIP material and 1μl of 1:10 dilution of total input were quantified by real-time PCR using primers amplifying the region from-1449 to -1342 of the PUMA promoter [3 (link)–4 (link)] (5′-TCCTCCTTGCCTGGGCTAG-3′ and 5′-GCGGACAAGTCAGGACTTGC-3′) or those amplifying the region from -1463 to -1271 of the p21^WAF1 promoter (5′-GGGTCTGCTACTGTGTCCTC -3′and 5′-TTGGTGCAGCTACAATTACTG-3′). All the ChIP-qPCR data were normalized to the Input-qPCR data following Percent Input Method [44 (link)].

To verify the tau-specific effects of the SARK and HMW samples, we immunodepleted total tau using HT7 antibody (MN1000, Invitrogen). PureProteome Protein G Magnetic Beads (LSKMAGG02, Millipore Corp) were resuspended by vortexing and 25 µl of the suspended beads were used for each sample. Using a magnetic rack, the storage buffer was removed and discarded. Beads were washed three times with 50 µl PBS 1×, and then 50 µl of HT7 antibody was added. The bead-HT7 solution was incubated for 1 h at 4 °C with head-over-tail rotation. The antibody flow-through supernatant was removed using a magnetic rack, and the beads were washed two more times with PBS. Next, 75 µl of either SARK or HMW tau was incubated with antibody–bead complexes overnight at 4 °C with head-over-tail rotation. The next morning, tau-immunodepleted supernatant was collected using a magnetic rack and stored at − 80 °C until further use. Immunodepletion was repeated twice more on the collected supernatant to fully capture residual tau in the sample. Tau immunodepletion was confirmed by western blotting and tau seeding assay normalizing on total protein levels (Supplementary Fig. 4a-c).