Anti trim28

The U2OS, Wi38-VA13 and HEK293T cells were cultured in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and cultured in an incubator containing 5% CO2 at 37 ℃. Human full-length TRIM28 cDNA was cloned into pDEST27 (Invitrogen) with GST tag and Plenti-HAFL-puro with HA-Flag (HAFL) double tags.
Cells were infected with lentivirus corresponding to shRNAs or overexpression vectors and selected with puromycin. Sense and antisense DNA oligos designed according to siRNA sequences were synthesized and cloned into pLKO.1-GFP vector. The siRNA sequences are: siTRIM28-1, 5′-CCUggCUCUgUUCUCUgUCCU-3′; siTRIM28-2, 5′-CUgAgACCAAACCUgUgCUUA-3′; siLuci, 5′-CUUACGCUGAGUACUUCGA -3′.
Antibodies used in this study include: rabbit polyclonal anti-TRIM28 (Abcam), rabbit polyclonal anti-Flag (Abmart), mouse monoclonal anti-tubulin (Sigma), rabbit polyclonal anti-GAPDH (Abmart), rabbit polyclonal anti-53BP1 (Novus), rabbit polyclonal anti-GST (Abmart), mouse monoclonal anti-PML (Millipore), rabbit polyclonal anti-Histone H3 (Abcam), rabbit polyclonal anti-H3K9me3 (Abcam) and rabbit polyclonal anti-SETDB1 (Proteintech).

Total protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitor cocktail, EDTA-free, 100X (APT006 or APT008, AntiProtech Inc.) Western blot analysis was performed using standard procedures as previously described [18 (link)]. The antibodies used were as follows: anti-NEK9 (cat. no. ab138488, Abcam), anti-ROBO1 (cat. no. 20219-1-AP, ProteinTech Group, Inc.), anti-Flag (cat. no. MA5558, AntiProtech Inc.), anti-STAT3 (cat. no. 12640, Cell Signaling Technology, Inc.), anti-p-STAT3 (cat. no. 9145, Cell Signaling Technology, Inc), anti-mouse IgG H&L (Alexa Fluor 488) (cat. no. ab150113, Abcam), anti-TRIM28 (cat. no. 15202-1-AP, ProteinTech Group, Inc.), anti-CTTN (cat. no. sc-55579, Santa Cruz Biotechnology, Inc.) anti-pCTTN (cat. no. 4569, Cell Signaling Technology, Inc.), anti-p100 (cat. no. 4882, Cell Signaling Technology, Inc.), anti-HA (cat. no. 51064-2-AP, ProteinTech Group, Inc.), anti-p-Ser (cat. no. sc-81514, Santa Cruz Biotechnology, Inc.) and anti-β-actin (cat. no. A2228, clone AC-74, Millipore Sigma).

For western blotting, samples in SDS sample buffer were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes prior to blocking in TBST with 5% (w/v) milk powder or 3% (w/v) bovine serum albumin and probing with primary and secondary antibodies (GE Healthcare). Primary antibodies were: anti-Myc (Sigma, #C3956), anti-FLAG (Sigma, #F3165), anti-HUWE1 (Novus Biologicals, #NB 100-652), anti-PCF11 (Bethyl Laboratories, #A303-706A), anti-GAPDH (Cell Signaling Technology, #2118), anti-TRIM28 (Abcam, #ab22553), anti-AMPKα1 (Cell Signaling Technology, #2795), and anti-MMS19 (Proteintech, #16015-1-AP) and Tb-anti-GST (Invitrogen, #PV3551). Secondary antibodies were: Donkey Anti-Rabbit IgG (GE Healthcare, NA934V) and Sheep Anti-Mouse IgG (GE Healthcare, NA931V). FLAG and TRIM28 primary antibodies were used at a dilution of 1:10,000 and Tb-anti-GST antibody was used at a dilution 1:720 and other primary antibodies were used at a dilution of 1:1000. All GE Healthcare secondary antibodies were used at a dilution of 1:5000. Protein signal was visualized after addition of ECL detection reagent (GE Healthcare) according to manufacturer’s instructions.