Protein g magnetic beads

Co‐IP was used to detect the interaction between DDX3X and G3BP1 and between NLRP3 and DDX3X as described previously.
⁴¹ (link) After harvesting tissue samples, total extracts were prepared by brief sonication in a Cell Lysis Buffer (Cell Signaling Technology, 9803). Primary rabbit anti‐G3BP1 (Proteintech, 13,057‐2‐AP), rabbit anti‐DDX3X (Proteintech, 11,115‐1‐AP), rabbit anti‐NLRP3 (Abcam, ab263899), and rabbit anti‐IgG (Cell Signaling Technology, 3900) antibodies were added to 200 μL cell lysate and incubated overnight at 4°C. Lysate and antibody (immunocomplex) solution was transferred to the tube containing protein G magnetic beads (Cell Signaling Technology, 70,024), incubated with rotation for 20 min at room temperature. A Magnetic Separation Rack (Cell Signaling Technology, 7017) was used to separate the magnetic beads according to the manufacturer's instructions and then boiled to denature the protein–bead complex. The immunoprecipitates from cells were subjected to a Western blot analysis.

Primary antibody against B cell leukemia/lymphoma 2 (Bcl-2) was obtained from Boster (Wuhan, Hubei, China). Primary antibodies against polyclonal antibodies against proliferating cell nuclear antigen (PCNA), Bcl-2 associated X (Bax), survivin, matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), β-Actin and normal rabbit or mouse immunoglobulin G (IgG) were obtained from Proteintech (Wuhan, Hubei, China). Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Primary antibodies against P38/p-P38 MAPK, JNK/p-JNK, ERK/p-ERK were obtained from Cell Signaling Technology (Danvers, MA, United States). HRP-conjugated anti-Rabbit IgG and HRP-conjugated anti-mouse IgG were obtained from Servicebio (Wuhan, Hubei, China). HRP-conjugated anti-Rabbit IgG light chain and HRP-conjugated anti-mouse IgG light chain were obtained from Abbkine Scientific (Redlands, CA, United States). Protein G magnetic beads were obtained from Cell Signaling Technology (Danvers, MA, United States). U0126, SP600125 and SB203580 were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Secondary follicles or ovaries (from 1, 2, 4, 6 or 10-day-old mice) were lysed with RIPA lysis buffer (Santa Cruz Biotechnology Inc). Proteins (150 μg) were treated with primary antibody (1:50 anti-ZP3, Proteintech) overnight at 4 °C. The mixture was further incubated with 10 µl of Protein G Magnetic Beads (Cell Signaling Technology, Tokyo, Japan) for 30 min at 4 °C and then washed 5 times in RIPA lysate buffer. The precipitates were suspended in 15 μl of 2 × SDS sample buffer. These were separated by SDS polyacrylamide gel (10% (v/v)) electrophoresis and transferred to PVDF membranes (GE Bioscience). The membranes were blocked in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris (pH 7.5), 150 mM NaCl and 0.05% Tween 20) containing 5% (w/v) BSA (Nacalai Chemical Co.). The blots were incubated with primary antibody (1:500 anti-pan cadherin) overnight at 4 °C. After washing in TBST, enhanced chemiluminescence (ECL) detection was performed by using the ECL system according to the manufacturer’s specifications (GE Bioscience) and appropriately exposing the blots to Fuji X-ray film (Fujifilm).

All Plasmids were generated by standard cloning procedures. Expression plasmids for 9myc-tagged Sox10 were based on pCMV5 and pBI-EGFP. Reporter plasmids for transgenic animals were generated by cloning the respective ECR fragments upstream of an Hsp68 minimal promoter followed by a lacZ cassette [38 (link)]. For luciferase assays the respective ECR fragments were cloned upstream of a β-globin minimal promoter followed by a luciferase cassette [22 (link)], Sox binding sites were mutated using the QuikChange XL site-directed mutagenesis kit (Stratagene). Mouse Neuro2a neuroblastoma cells and rat primary oligodendroglia were kept in culture as described [39 (link)]. Transient transfections of Neuro2a cells, luciferase assays and EMSA followed standard procedures [22 (link)]. ChIP was performed as reported [31 (link)] with the following modifications: Chromatin was prepared from primary oligodendrocytes kept under differentiating conditions for six days and from brain tissue of three week old mice. Fixation was with 1% formaldehyde in PBS. For precipitation of sheared chromatin, anti-Sox10 antiserum and corresponding preimmune serum were used in combination with protein G magnetic beads (Cell Signaling Technology). A list of primers for cloning and detection of genomic fragments in PCR experiments, including their sequence and position is available upon request.

Telomeric ChIP was performed as previously described (Loayza and De Lange, 2003 (link)). Telomeric DNA associated with shelterin proteins was immunoprecipitated with the following crude sera or purified antibodies: crude rabbit TRF1 (#371), crude rabbit TIN2 (#865), crude rabbit TPP1 (#1151), POT1 (Abcam, ab123784), anti-HA (Abcam, ab9110) and protein G magnetic beads (Cell signaling). For ChIP of exogenously introduced TIN2 alleles, 293T cells were transfected by calcium phosphate transfection, and crosslinked and harvested 36–48 hr after transfection.

Cells were harvested and cross-linked with 37% paraformaldehyde for 10 min. After incubation with 0.125 M glycine to terminate the cross-linking, the cells were lysed using a lysis buffer (1 M Dithiothreitol, 200× Protease Inhibitor Cocktail), followed by incubation for 10 min on ice. After centrifugation at 2000×g, the nuclei of the cells were digested in 0.5 μl Micrococcal Nuclease and sonicated to shear the DNA to a size range of 150–900 bp. Then, 30 μl Protein G Magnetic Beads (Cell Signaling Technology, United States) were added to the mixture. After washes with cold high/low salt lotion, the mixture was incubated with anti-NFIB antibody (1:30, #ab186738, Abcam) or control immune-globulin G (IgG) overnight at 4°C. Subsequently, the beads were eluted with elution buffer (100 mM NaHCO3 and 1% SDS). The eluate was incubated with 150 μl 1× ChIP at 65°C for 30 min and then with a buffer (6 μl 5 M NaCl, proteinase K, pH 8.0) at 65°C for 2 h, followed by DNA purification with a centrifugal column at 18,500 × g for 30 s.