Bsa assay kit

Isolation of crude membrane and soluble fractions was performed as described previously (Distler et al., 2014b (link)). All steps were performed at 4°C. Briefly, the striatum was mechanically homogenized with a motorized tissue grinder in lysis buffer [5 mM HEPES, 0.32 M sucrose, 1× protease and phosphatase inhibitor cocktail (Thermo Fisher), pH 7.5] at a weight:volume ratio of 1:10 (1 mg per 10 μL). Homogenates were centrifuged at 1,000× g for 10 min. Following removal of the pellet (P1), the supernatants were collected and centrifuged again at 12,000× g for 10 min. The soluble protein fraction was collected from the supernatant, and the resulting pellet (P2) was collected as the crude membrane and mitochondrial enriched protein fraction and resuspended in PBS. Fractions were stored at −80°C prior to processing for proteomics analysis. Protein levels in extracts were quantified using a BSA Assay Kit (Thermo Scientific) on the Agilent Nanodrop device. Note that the P2 was retrieved from each individual mouse and samples were not pooled for the proteomics analysis described below.

Total protein from each group of cells was isolated with 20 µL RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) supplemented with inhibitor cocktail (Thermo Fisher Scientific; Waltham, MA, USA), phenylmethyl sulfonyl fluoride (PMSF; Enzo) and sodium orthovanadate (Santa Cruz Biotechnology). The extracted proteins were quantified using BSA assay kit (Thermo Fisher Scientific, IL, USA), and the resulting absorbance value was measured with an ELISA microplate reader (Thermo Fisher Scientific) at 562 nm. An equivalent amount of proteins was loaded in a 12% SDS-polyacrylamide gel, and was separated by electrophoresis. The proteins were then electrically transferred onto a PVDF transfer membrane (GE Healthcare, Buckinghamshire, UK). The membrane was blocked using 5% normal horse serum in TBS buffer containing 0.1% Tween 20 (TBS-T) for 90 min. After blocking, the membrane was washed, and pan-cytokeratin primary antibody (dilution 1:200; Santa Cruz Biotechnology) was added and incubated in 4 °C overnight. The membrane was washed again with TBS-T buffer, and horseradish peroxidase conjugated antimouse secondary antibody (dilution 1:2000; Santa Cruz Biotechnology) was added and incubated for 90 min at room temperature. The protein expression was detected using enhanced chemiluminescence (ECL) detection system (Amersham) and exposed to X-ray film (Agfa).

HUTK-143B cells were infected with VV-iPDL1/GM at MOI = 2 without FBS. Culture media was collected 48 h post infection, and filtered by 0.8 μm syringe filter unit (Millipore, Darmstadt, Germany). The media was incubated with 200 μL Protein G Sepharose (Sigma-Aldrich, St Louis, MO) at 4 °C overnight. The protein G beads were washed by 1× PBS three times, and eluted by 0.1 M glycine-HCL, pH=2.8. The elution was dialyzed in 4 L 1 × PBS overnight^{30 (link),31 (link),60 (link)}. The concentration of the iPDL1 protein was determined using BSA Assay kit (Thermo, Waltham, MA).

Total proteins were isolated from the cells using 50 µL RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with the addition of inhibitor cocktail and phenylmethylsulfonyl fluoride for 30 min on ice and then centrifuged at 16,000× g for 20 min. The protein concentration was measured using a BSA assay kit (Thermo Fisher Scientific; Waltham, MA, USA). Using the isolated proteins, an immunoblot assay was performed using appropriate primary antibodies against NMDAε2 (1:200), p53 (1:200), CREB (1:500), p-CREB (1:500), GAPDH (1:1000) (Santa Cruz Biotechnology), and caspase-3, active (cleaved) form (1:150) (Merck Millipore, Temecula, CA, USA) and incubated at 4 °C overnight. Thereafter, horseradish peroxidase-conjugated anti-goat, anti-rabbit, and anti-rabbit secondary antibodies were used (Santa Cruz Biotechnology). Protein bands were captured on X-ray films using the GE ECL detection system.