Normal mouse igg antibody

Chromatin immunoprecipitations were performed using HFKs, CIN612 cells, or stable HFK 31 cells in the presence or absence of HU. Cells were cultured as described above. Chromatin immunoprecipitation was performed using γ-H2AX (Millipore; catalog no. 05-636), RAD51 (Millipore; catalog no. 05-530-I), BRCA1 (Thermo Fisher; catalog no. MA1-23164), SMC-1 (Abcam, Inc.; catalog no. ab9262), and normal mouse IgG antibody (Santa Cruz; catalog no. SC-2025). Protocol was performed as previously described (37 (link)).
Real-time PCR was performed with a LightCycler 480 system (Roche) using HPV 31 URR primers, Alu primers, or HPV 31 L2 primers as follows: URR Forward, 5′-AAC|TGC|CAA|GGT|TGT|GTC|ATG|C-3′; URR Reverse, 5′-TGG|CGT|CTG|TAG|GTT|TGC|AC-3′; Alu Forward, 5′-ACG|AGG|TCA|GGA|GAT|CGA|GA-3′; Alu Reverse, 5′-CTC|AGC|CTC|CCA|AGT|AGC|TG-3′; L2 Forward, 5′-TTT|GGT|GGG|TTG|GGT|ATT|GG-3′; L2 Reverse, 5′-GTA|GGA|GGC|TGC|AAT|ACA|GAT|G-3′. All primers were determined to have similar levels of efficiency based on the methods previously described by Livak et al. (38 (link)).

Immunoprecipitation and immunoblot analysis were performed as described previously^{35 (link)}. Briefly, the cells were treated as indicated and washed with cold PBS. The cells were harvested with lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0% Triton X-100, 1 mM EDTA, and 10% glycerol containing protease inhibitors). Cell extracts were left on ice for 30 min and centrifuged at 13,000 r.p.m for 30 min. Cell lysates were incubated with 2μg of anti-RIPK1 antibody or 2μg of normal mouse IgG antibody (Santa Cruz) overnight at 4°C and then incubated with 15μl of protein A beads for an additional 3h at 4°C. The beads were washed with lysis buffer three times and then boiled in 20μl of 1×SDS loading buffer for 10 min. Samples were loaded on SDS-PAGE gels followed by electroblotting onto PVDF membranes (Millipore). To determine protein levels, immunoblot was performed using the following antibodies: anti-RIPK3 (2283) from Pro Sci; anti-pMLKL (ab196436) from Abcam; anti-RIPK1 (610459) from BD Biosciences; anti-pP38 (9211S), anti-JNK (9252), anti-pERK1/2 (4370), anti-pRIPK1 (S166) (31122S), anti-pJNK (9251) from Cell Signaling, anti-Tim23 (11123-1-AP) from Proteintech, anti-GAPDH (sc-32233) from Santa Cruz, and anti-TRIF (657102) from Biolegend.

Cells were grown to approximately 90% confluency, and protein-DNA complexes were cross-linked with 1% formaldehyde for 10 min followed by the addition of 0.125 M glycine for 10 min. The cells were harvested, lysed in lysis buffer [1% NP-40, 0.1% SDS, 5 mM EDTA, 150 mM NaCl, 0.5% deoxycholate, 50 mM Tris, pH 8.0, 2% cOmplete Protease Inhibitor Cocktails (Roche)], and sonicated to lengths between 400 and 1000 bp. The crosslinked, sonicated chromatin was precleared with 10 μL of Dynabeads protein G (Invitrogen) before incubation with 2 μg of the indicated antibodies and rotated at 4°C overnight. Dynabeads protein G were added for an additional 15 min. Normal mouse IgG antibody (Santa Cruz Biotechnology) was used as the control immunoprecipitation. After extensive washes, immunocomplexes were treated with Proteinase K and decrosslinked at 65°C for 6 h. Bound DNA in the ChIP was extracted by the PCR purification kit (Qiagen, Chatsworth, CA, USA) and subjected to PCR analysis using primers that were designed to span the Dp1 and E2F1 binding sites (Kpna2 F: ATGGGCACACAGCTTAG; Kpna2 R: CTGAGTCTGTACCTGCGAA). After amplification, PCR products were separated on a 2% agarose gel and were analyzed under UV light to view the stained DNA.

Cell lysates (500 μg) were pre-cleared with 30 μl of protein A/G plus-agarose beads (Santa Cruz, catalog #: sc-2003) and 1 μg of normal mouse IgG antibody (Santa Cruz, catalog #: sc-2025) for 1 h at 4°C to remove nonspecific binding proteins. The pre-cleared cell lysates were incubated with 2.0 μg of mouse anti-α1 antibody for 1 h at 4°C, and then with 30 μl of protein A/G plus agarose beads overnight at 4°C. For FLAG-tagged proteins, the pre-cleared cell lysates were incubated with 30 μl of anti-FLAG M2 magnetic beads (Sigma, catalog #: M8823-5 mL) overnight at 4°C. IgG serves as negative control. The beads were collected by centrifugation at 8000 × g for 30 s or using a magnet separator (Promega), and washed three times with lysis buffer. The complex was eluted by incubation with 30 μl of Laemmli sample loading buffer in the presence of β-mercaptoethanol. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed.