Anti igg antibodies

AGEs were added to HepG2 cells cultured in DMEM supplemented with 0 mM or 25 mM glucose and cells were cultured for another 24 hours. Nuclear and cytosolic fractionation and western blot analysis was performed as described previously.^{[15 (link)]} The following primary and secondary antibodies were used: anti-ChREBP (Novus), and anti-Tubulin (Sigma) antibodies, and secondary peroxidase labeled anti-IgG antibodies (Santa Cruz)

The 3′UTR region sequence of human GRN (322 pb) was amplified by RT-PCR from SK-N-BE total RNA using the primers listed in Supplementary Table S2, cloned into the TOPO TA vector (Thermo Fisher Scientific) downstream of the T7 promoter and linearized (0.5 μg) by HindIII restriction enzyme (30 U) for in vitro transcription. The mouse Grn 3′UTR probe [7 (link)] was used as a positive control. UV-crosslinking was performed using HEK293T protein lysates (200 μg) transfected with human Flag-TDP-43 and ³²P-radiolabeled riboprobes, as previously described [46 (link)]. Immunoprecipitation was conducted on UV-crosslinked samples using the anti-FLAG (2 μg; Sigma-Aldrich) or the anti-IgG antibodies (2 μg, SantaCruz Biotechnology) (Supplementary Table S1) and protein G Dynabeads (Thermo Fisher Scientific). Immunocomplexes were washed several times in PBS with 0.02% Tween-20, run on a 10% SDS-PAGE gel and analyzed by autoradiography.

Western blot analysis was performed as previously described (17 (link)). Total protein was isolated from PBMCs using TRIzol Reagent (British Biocell International). Proteins were quantified by a Bradford assay. Total protein (50 μg) was separated on 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) gels, and electro-transferred to NC membranes (Millipore, Bedford, MA, USA). After non-specific binding sites were blocked with 5% skim milk for 1 h, the membranes were then incubated in a mixture of Tris-buffered saline and Tween-20 (TBS-T) containing survivin (1:1,000), CD4 (1:100), CD8 (1:100), CD44 (1:100), VEGF (1:200) and ICAM-1 (1:200) antibodies for 2 h at room temperature. After washing, the membrane was incubated in TBS-T containing the appropriate secondary anti-IgG antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The target protein was detected using western blotting luminol reagent (Santa Cruz Biotechnology, Inc.), according to the manufacturer’s instructions. Results were visualized by autoradiography using X-Omat autoradiography film (Kodak, Rochester, NY, USA). For protein loading normalization, a similar procedure was performed using a monoclonal antibody against tubulin (1:1,000; Beyotime Institute of Biotechnology, Hangzhou, China) as an internal standard. Three replicates were performed for each sample.