Anti gfp antibody

HEK293 cells co-expressing EGFP-PKCδ and HA-ARTD10-WT were treated with 3 μM OUL35 or the appropriate volume of DMSO as vehicle control for 16 h. Forty-eight hours post transfection, cells were lysed in RIPA buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 1% DOC; 0.1% SDS; protease inhibitor cocktail, Sigma) including 10 μM Olaparib (Selleckchem) and the lysates were cleared at 4 °C for 30 min. EGFP-PKCδ was immunoprecipitated with 5 μl of GFP-TRAP magnetic agarose beads (Chromotek) at 4 °C for 1 h. Afterwards, the beads were washed three times in RIPA buffer. Samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes to visualize MARylation using an anti-PAR/MAR antibody (Cell Signaling Technology, #83732). The individual proteins were analyzed using anti-GFP antibodies (Rockland, mouse monoclonal 600-301-215 M) to visualize EGFP-PKCδ, anti-α-Tubulin (Sigma, #T5168), anti-ARTD10 [6 (link)], goat-anti-rabbit-HRP (Jackson Immunoresearch, #111-035-144), and goat-anti-mouse-HRP (Jackson Immunoresearch, #115-036-068).

For heterochromatin staining we used antibodies for H3K9me2 (ab1220, concentration of 1:400), H3K27me3 (ab205728, 1:50) H4K20me1 (ab9051, 1:500). For euchromatin markers we used antibodies for H3K27ac (8173s, 1:100) and H3K4me3(11960s, 1:50). Active transcription was measured using an antibody for RNA polymerase II CTD repeat YSPTSPS phospho S5 (ab5131, 1:100). To track retroviral activity and transcription regulation loss we used antibodies for L1-ORF1p (ab216324, 1:100), and Dicer (ab167444, 1:200). GFP expression was measured using anti-GFP antibody (Rockland 600-101-215, 1:250).

For all SMOR experiments, 24 × 50 mm high precision glass coverslips (Bioscience Tools) and drilled microscope slides were passivated with a combination of PEG and PEG-biotin (coverslips) or PEG only (slides) following a previously published protocol for SiMPull^{31 (link)}. Reaction chambers (~20 µl) were created using double-sided tape and epoxy. Protein was immobilized on the surface with 0.2 mg ml⁻¹ NeutrAvidin in buffer C (20 mM HEPES pH 7.5, 150 mM KCl, 10% glycerol, 0.05% Tween-20, 1 mM DTT). Excess NeutrAvidin was washed away and incubated with either biotin-conjugated mouse monoclonal anti-Strep-Tag II antibody (LSBio, Cat. No. LS-C203632-100) or biotin-conjugated rabbit polyclonal anti-GFP antibody (Rockland, Cat. No. 600-406-215) diluted in buffer C + BSA (0.1 mg ml⁻¹; Molecular Biology Grade Bovine Serum Albumin). Activator WD40 and activated APC/C (typically 50 µl containing about 65 ng APC/C, of which only a small fraction is immobilized on the glass) were immobilized using anti-Strep-Tag II, while APC/C alone was immobilized with anti-GFP. SMOR results were similar when performed in the buffer (wash buffer B) used in polarization anisotropy.

All steps of cell lysis and immunoprecipitation were performed at 4°C using ice cold buffers. Cells were lysed on the dish with 1x Lämmli buffer. Proteins were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (PALL) and probed with the respective antibodies. Primary antibodies against PLCγ and phosphospecific antibodies against STAT3 (pTyr705), ERK1/2 (pThr202/pTyr204), PDGFRα(pTyr849)/β(pTyr857), AKT (pSer473) and the HA-tag (6E2) were purchased from cell signaling. Anti-STAT1 and anti-STAT3 antibodies and phosphospecific antibodies for STAT1 (pTyr701), STAT5 (pTyr694) and PLCγ1 (pTyr783) were purchase from BD. Antibodies against STAT5 (C-17), PDGFRα (C-20), ERK1 (K-23), AKT1/2 (N-19) and tubulin (DM1A) were purchased from Santa Cruz Biotechnologies. Anti-GFP antibody was obtained from Rockland. The horseradish peroxidase-conjugated secondary antibodies were purchased from Dako (anti-goat Ab) or cell signalling (anti-rabbit and anti-mouse Abs). Signals were detected using an ECL solution containing 2.5 mM luminol, 2.6 mM hydrogenperoxide, 100 mM Tris/HCl pH 8.8 and 0.2 mM para-coumaric acid [54 (link)].

Protein lysates were obtained from control versus nephrin morphants and from adriamycin non-exposed versus exposed zebrafish larvae. Blots were incubated with a rabbit polyclonal anti-PACAP antibody (produced in our laboratory, as previously described) [16 (link)], a rabbit monoclonal anti-ceruloplasmin antibody (Dako, Glostrup, Denmark), a goat polyclonal anti-GFP antibody (Rockland Immunochemicals, Gilbertsville, USA) and a rabbit monoclonal anti-β-actin antibody (Cell Signaling Technology, Boston, USA). Secondary anti-rabbit and anti-goat antibodies were from Dako. Blots were stained with Western blotting electrochemiluminescence (ECL) detection reagent (Thermo Scientific, Rockford, USA). Detection of signal and measuring of band intensity were performed with ImageJ software. The band intensities of PACAP, ceruloplasmin, and GFP were corrected for the band intensities of the loading control β-actin [26 (link)] and were expressed as a percentage of the control condition. All Western blots were performed at multiple time points during the first 6 days of development and at least in duplicate.