Anti flag m2

HEK293T/17 cells were obtained from the American Type Culture Collection (ATCC). The HEK293T 5XUAS-Luciferase cell line has been previously described (Wysocka et al., 2005 (link)). V6.5 mouse embryonic stem cells were grown on 0.2% gelatinized plates in Knockout DMEM supplemented with 15% FBS, 1% Glutamax (35050; Invitrogen, Carlsbad, CA), 1% nonessential amino acids, 1% Pen/Strep, 0.2% β-mercaptoethanol, LIF (ESGRO1107; 1:10000; Millipore, Billerica, MA), and 2 µg/ml doxycycline (D9891; Sigma, St. Louis, MO). To maintain consistent levels of doxycycline, media was changed every 2 days. Antibodies used were: anti-FLAG-M2 (F1804; Sigma, St. Louis, MO), anti-β actin (ab8227; Abcam, Cambridge, MA), anti-RbBP5 (A300-109A; Bethyl, Montgomery, TX), anti-H3K4me3 (ab8580; Abcam, Cambridge, MA), anti-β tubulin (ab6046; Abcam, Cambridge, MA), anti-H3 (ab1791; Abcam, Cambridge, MA), anti-WDR5 (07-706; Millipore, Billerica, MA), and anti-HA (MMS-101P; Covance, Princeton, NJ). All immunoprecipitations were conducted using anti-FLAG-M2 (A2220; Sigma, St. Louis, MO) or mouse IgG (A0919; Sigma, St. Louis, MO) agarose beads. Unless noted, all expression vectors were cloned into pcDNA3 or pcDNA3.1 + backbones.

293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). RNase inhibitors were omitted from samples treated with 15 μg/ml RNaseA (Life Technologies). FLAG- or T7-tagged L1 proteins were immunoprecipitated with anti-FLAG M2 (Sigma) or anti-T7 (Novagen) monoclonal antibodies coupled to magnetic beads (Dynabeads, Thermo Fisher). Precipitated proteins were separated by SDS page and transferred onto PVDF membranes and probed with anti-myc (9B11) antibody (Cell Signaling) anti-FLAG M2 (Sigma), or anti-T7 antibody (Novagen). Next, membranes were incubated with anti-mouse HRP light chain-specific or anti-rabbit HRP conformation-specific secondary antibodies (Cell Signaling). For immunoprecipitation of SAMHD1 combined with subsequent LEAP reaction (LEAP-IP), 293T cells transfected with pAD2TE1 and SAMHD1-myc or empty vector were lysed and myc-tagged SAMHD1 was precipitated using anti-myc antibody bound to magnetic beads (Cell Signaling). The beads harboring SAMHD1 and L1 RNPs were eluted in MLV-RT buffer (Promega). Samples for LEAP reaction were directly subjected to reaction, samples for MLV-RT reactions were incubated for 10 min at 95 °C. LEAP and MLV-RT reactions were performed as described above.

HEK293T cells were transfected 48 h before co-immunoprecipitation assay with Pires-hr-1a encoding 3 × Flag-SEMG1 and 3 × Flag-SEMG2. Control cells were transfected with native vector. Cells were lysed in the hypotonic buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM EDTA, 1 mM NaF 0.25%, Triton X-100 and protease inhibitor cocktail) on ice during 10 minutes. Next, 150 mM NaCl was added to increase the ionic strength of the buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0,1%, Triton X-100 and protease inhibitor cocktail) with additional 10 min of lysis on ice. Cell lysates were cleared by centrifugation and were then incubated with anti-Flag M2 (Sigma, USA) agarose beads for 4 h followed by three washing steps with TBS buffer. Recovered immune complexes were eluted by boiling in Laemmli buffer and were subjected to western blot analysis.
For reciprocal co-immunoprecipitation, H520 cells expressing both SEMGs were used. These cells were transiently transfected by pCDH vector encoding 3 × Flag-tagged LDHA, PKM2 or empty vehicle. Lipofectamine 2000 was used as transfecting reagent (according to manufacturer’s recommendation). Co-immunoprecipitation of endogenous proteins was carried out 48 h post-transfection using anti-Flag M2 (Sigma, USA) agarose beads as was described for HEK293T cells.

Formaldehyde- or paraformaldehyde-fixed tumor tissues were embedded in paraffin, and sections were stained with H&E using standard techniques. The following primary antibodies were used: anti-FLAG M2 (Sigma-Aldrich), anti-Sox9 (Sigma-Aldrich), anti-Hey1 (Abcam), anti-Runx2 (MBL), anti-CK18 (Proteintech), anti-Galectin 3 (Proteintech), anti-Ki-67 (Abcam), and anti-NCOA2/SRC2 (Cell Signaling).
Immunoblotting was performed using whole-cell lysates. The following primary antibodies were used: anti-FLAG M2 (Sigma-Aldrich), anti-Runx2 (MBL), anti-Runx3 (Abcam), anti–α-tubulin (Sigma-Aldrich), and anti-Cas9 (Novus, Centennial, CO).