Control igg antibody

Cultured cortical neurons (at DIV11) were cross-linked with 1% (v/v) formaldehyde, followed by adding 125 mM of glycine to quench the cross-linking reaction. The cells were lysed in SDS lysis buffer (1% SDS, 1 mM EDTA, and 50 mM Tris–HCl, pH 8.0) including protease inhibitors and phosphatase inhibitors. Lysate was sonicated to produce chromatin fragments of 200–500 bp. Sheared chromatin was immunoprecipitated with Dynabead (Thermo Fisher Scientific, Inc., Waltham, MA, United States, Cat# 10003D), conjugated with 1 ug of H3K4me3 (Abcam, Cambridge, United Kingdom, Cat# ab8580), H3K27me3 (Merck, Rahway, NJ, United States, Cat# 07–449)-specific antibodies, or control IgG antibodies (Cell Signaling Technology, Inc., Danvers, MA, United States, Cat# 2729) overnight at 4°C with rotation. After reverse crosslinking, DNA was purified by using phenol-chloroform and ethanol precipitation.

A ChIP assay was performed using a Chromatin Immunoprecipitation Kit (Millipore, MN). Briefly, the cells were collected and crosslinked with 1% (v/v) formaldehyde for 10 min at room temperature. Nuclei were subsequently isolated via the lysis of the cytoplasmic fraction, and the chromatin was divided into 150–900 bp fragments via ultrasonic disruption using a standard microtip and a Branson W250D Sonifier (four pulses, 60% amplitude, duty cycle 40%). The sonicated nuclear fractions were divided for input control and incubated at 4 °C overnight with anti-Stat3 antibodies (Cell Signaling Technology, MA, USA) or control IgG antibodies (Cell Signaling Technology, MA). After incubating with 30 ml of ChIP grade protein A/G-agarose beads for 2 h at 4 °C, the antibody-protein-DNA complexes were eluted from the beads and digested using Proteinase K (40 mg) for 2 h at 65 °C, followed by spin column-based DNA purification. Finally, the genomic DNA recovered from the ChIP assays was amplified via qPCR with primers () specific for the Stat3 binding region of the CD147 promoter. The specificity of each primer set was verified by an analysis of the dissociation curve of each gene-specific PCR product.

HeLa cells were treated as described in Figure legends and lysed in lysis buffer (50mM Hepes, 50mM NaCl, 10% glycerol, 1% Tryton X-100, 1.5mM MgCl, 5mM EGTA) for 15 min on ice and pelleted for 10min at 13.000 rpm. The supernatant was incubated with the WIPI2 antibody (8567 Cell Signaling) or control IgG antibodies (2729S Cell Signaling) (1:100) for 3 h and 2 h with Dynabeads Protein A (Novex-Lifechnologies). The immunoprecipitate was eluted by boiling the samples in Laemmli buffer for 5 min. GFP-tagged proteins (GFP-WIPI2, GFP-RAB11A) were pulled down using GFP-TRAP beads (ChromoTek) according to the manufacturer’s protocol. Proteins were resolved by SDS-PAGE.