Benchmark pre stained protein ladder

293-ORF6 [21 (link)] cells in 60 mm dishes were infected with HuAd5 null, HuAd5-PyE140na, or HuAd5-PyE140co at a multiplicity of infection (MOI) of either 500 or 6,500 pu/cell or transfected with 8 μg of DNA-PyE140na using lipofectamine 2000CD (Invitrogen). Cell lysates were harvested in SDS protein gel loading solution (Quality Biological Inc., Gaithersburg, MD) 24 hours or 48 hours post-infection/transfection, run on a 4–20% Tris-Glycine acrylamide gel (Invitrogen) and transferred to an Immobilon-P PVDF membrane (Millipore Corp., Bedford, MA). The primary antibody was a 1:100 dilution of sera from mice immunized with DNA-PyE140na and HuAd5-PyE140na. The secondary antibody was a 1:2,500 dilution of polyclonal goat anti-mouse IgG + IgM conjugated to alkaline phosphatase (Applied Biosystems Inc., Foster City, CA). The marker is BenchMark Prestained Protein Ladder (Invitrogen). Proteins were visualized with an alkaline phosphatase Western-Light Chemiluminescent Detection System (Tropix Inc., Bedford, MA) and an alkaline phosphatase colorimetric substrate (KPL Inc., Gaithersburg, MD).

Immunoblot (IB) experiments were performed as described previously (Jones et al., 2004 (link), 2007 (link); Balut et al., 2010a (link),b (link); Gao et al., 2010 (link); Bertuccio et al., 2014 (link); Farquhar et al., 2017 (link)). Briefly, cells were lysed and protein concentrations were determined by the BCA protein assay (Walker, 1994 (link)). Equal amounts of protein (30 μg) were loaded into wells of a gel (6 or 8%) and protein standard (8 μl) used (BenchMark™ pre-stained protein ladder; Invitrogen, Cat No. 10748-010) and resolved with SDS-PAGE for 150 mV for 90 min (Hoefer Mighty Small II system, Cat. No. 80-6149-35, Amersham Biosciences Corp. Piscataway, NJ, USA). Proteins were transferred (50 V, 2 h) with a semi-dry transfer unit (Hoefer, EPS 2A200) to polyvinylidene difluoride (PVDF) membranes for further IB analysis with α-streptavidin antibody. Proteins bands were visualized by enhanced chemiluminescence detection (Lumilight, Roche, Basel Switzerland). Blots were probed for β-actin as a protein loading control. The bands obtained from immunoblot analysis were quantified by densitometry, using the GS-700 densitometer (Bio-Rad) and the Quantity One programme (BioRad laboratories). The obtained band intensities for the various time points were normalized to β-actin and then compared relative to the intensity at time 0 (t = 0) and reported.

The protein concentration was determined using the Bradford method. 30 μg of cell lysates (1 % Nonidet buffer) were boiled for 10 min at 60 °C in Laemmli sample buffer. After that samples were loaded on 12 % SDS-PAGE polyacrylamide gel and separated. After protein transfer to PVDF membranes (BioRad), membranes were blocked for 1 h with 5 % milk at 4 °C and incubated with primary antibodies diluted in blocking solution overnight (4 °C). Primary antibodies (Abcam 126534) were then detected with HRP-conjugated secondary antibodies and chemiluminescence was triggered by Western Bright ECL reagent (Advansta) as substrate, and recorded on CL-XPosure films (Pierce). Blots and films were scanned. Molecular weight of protein bands was estimated in comparison to BenchMark Prestained protein ladder (Invitrogen).

M-PER Mammalian Protein Extraction Reagent (Cat#78503, Thermo Fisher Scientific, Waltham, MA) together with Proteinase Inhibitor Cocktail (Cat#P8340, Sigma Aldrich, St. Louis, MO) were used to extract proteins from MDA_w, MDA_KDRab27a and MDA_KDTRAF3IP2 cells or from conditioned media of MDA_w (EXO_MDAw) or MDA_KDRab27a (EXO_MDAKDRab27a) cells. After gel electrophoresis of equal amounts of protein using 12% Precise Tris-Glycine Gels (Cat#0025267, Thermo Fisher Scientific), Laemmli Sample Buffer (Cat#161-0747, BioRad Laboratories, Hercules, CA) and BenchMark Pre-Stained Protein Ladder (Cat#10748-010, Invitrogen, Carlsbad, CA) the proteins were electroblotted and the following primary antibodies were used: GAPDH (0.0002 mg/ml; Cat#ab9485, Abcam, Cambridge, MA), Rab27a (0.01 mg/ml; Cat#sc-22756, Santa Cruz Biotechnology, Inc.), TRAF3IP2 (0.01 mg/ml; Cat#WH0010758M1-100UG, Sigma-Aldrich), CD9 (0.01 mg/ml; Cat#MA1-19002, Thermo Fisher Scientific), or MHCII (0.01 mg/ml; Cat#MA1-19143, Thermo Fisher Scientific). Goat Anti-Rabbit IgG-HRP (Cat#sc-2004, Santa Cruz Biotechnology, Inc.) or Donkey Anti-Mouse IgG-HRP (Cat#sc-2318, Santa Cruz Biotechnology, Inc.) served as secondary antibodies.

M-PER Mammalian Protein Extraction Reagent (Cat#78503, Thermo Fisher Scientific, Waltham, MA) and Proteinase Inhibitor Cocktail (Cat#P8340, Sigma-Aldrich, St. Louis, MO) were used to extract proteins from U87_TRAF3IP2KD, U87_{Control shRNA}, U118_TRAF3IP2KD, and U118_{Control shRNA} cells. After gel electrophoresis of equal amounts of protein using 12% Precise Tris–Glycine Gels (Cat#0025267, Thermo Fisher Scientific), Laemmli sample buffer (Cat#161-0747, Bio-Rad Laboratories, Hercules, CA), and BenchMark Pre-Stained Protein Ladder (Cat#10748-010, Invitrogen, Carlsbad, CA), the proteins were electroblotted and the following primary antibodies were used: GAPDH (0.0002 mg/ml; Cat#ab9485, Abcam, Cambridge, MA), TRAF3IP2 (0.01 mg/ml; Cat#WH0010758M1-100UG, Sigma-Aldrich), CD31 (0.01 mg/ml; Cat#PA5-16301, Invitrogen), IL1β (0.01 mg/ml; Cat#710331, Invitrogen), IL6 (0.01 mg/ml; Cat# MA5-23698, Invitrogen), IL8 (0.01 mg/ml; Cat# PA5-86028, Invitrogen). Goat Anti-Rabbit IgG-HRP (Cat#sc-2004, Santa Cruz Biotechnology, Inc.) or Donkey Anti-Mouse IgG-HRP (Cat#sc-2318, Santa Cruz Biotechnology, Inc.) served as secondary antibodies.