Anti gfp

For ubiquitination assays, HEK 293 T cells were transfected with the indicated plasmids using Lipofectamine 2000 for 6 h. Then, the media were replaced with normal culture media. After transfecting for 24 h, the cells were treated with 10 µM MG132 for 2 h before harvesting. The cells were then lysed using co-IP lysis buffer supplemented with 20 mM NEM (Sigma, Burlington, MA). The cell lysates were centrifuged at 15,000 rpm at 4 °C for 20 min. Among the same amount of protein in the supernatant by by bicinchoninic acid (BCA) analysis, 10% supernatant was immunoblotted to confirm each protein expression. The remaining supernatants were incubated with 1 mg anti-GFP (Santacruz, CA, USA) or anti-RFP antibodies overnight at 4 °C. The antibody-binding proteins were pulled down by incubation for 1 h at 4 °C with 20 µL Protein A or G magnetic beads (ThermoFisher, Waltham, MA). The beads were washed three times with co-immunoprecipitation lysis buffer and heated in 1 × SDS sample loading buffer. The eluted proteins were immunoblotted with anti-RFP, anti-GFP, anti-ubiquitin (Santacruz, CA), or anti-β-actin antibodies.

Lysates of SFs or extracts from synovial tissues were subjected to immunoblot with anti-p73 (Santa Cruz), anti-GFP (Santa Cruz), or anti-cadherin-11 (Cell Signaling), and the blots were re-probed with anti-β-actin (Sigma-Aldrich) as a quantitative control, as previously described [8 (link), 20 (link)]. AdLacZ or Ad37AA-transduced SF lysates were immunoprecitated with anti-iASPP (Santa Cruz), followed by immunoblotted with anti-p73 (Santa Cruz) or anti-GFP (Santa Cruz), and small quantities (10%) were snapped for immunoblot analysis as an input control.

Primary motoneurons or E18 spinal cord tissue, respectively, were lysed with cytosolic and nuclear fractionation buffer, solubilized in Laemmli buffer (125 mM Tris, pH 6.8, 4% SDS, 10% β-mercaptoethanol, 20% glycerol, and 0.004% bromophenol blue) and boiled for 10 min at 99°C. Proteins were then subjected to SDS-PAGE, blotted onto PVDF membrane, incubated with the corresponding antibodies, and developed with either ECL or ECL Advance Systems (GE Healthcare) on X-ray film (Fuji super RX). Western blots were scanned and quantified by densitometry analysis with ImageJ (National Institutes of Health). For Western Blot analysis the following primary and secondary antibodies were used: anti-SMN (BD Biosciences, 610646, 1∶3000), anti-hnRNP R (Abcam, ab30930, 1∶3000 or polypeptide antiserum aa1-18, ICN, Wuerzburg, 1∶3000), anti-GFP (Santa Cruz, sc-8334, 1∶4000), anti-GAPDH (Millipore, 6C5, 1∶4000), anti-α tubulin (T5168, Sigma, 1∶4000), anti-histone H3 (Abcam, ab8580, 1∶20 000), anti-calnexin (Abcam, ab22595, 1∶5000), anti-GFP (Santa Cruz, sc-8334, 1∶4000), anti-mouse IgG (Jackson Immunoresearch, 115-035-003, 1∶10000), anti-rabbit IgG (Jackson Immunoresearch, 111-035-003, 1∶10000), anti-mouse light chain-specific (Jackson Immunoresearch, 111-035-174, 1∶10000) and anti-rabbit light chain-specific (Jackson Immunoresearch, 211-032-171, 1∶10000).