M per extraction buffer

PDX tumor samples were snap frozen in liquid nitrogen and then pulverized using the Cellcrusher (Cork, Ireland). Protein lysates were prepared in M-PER extraction buffer (Thermo Scientific, Waltham, MA) containing a protease inhibitor cocktail (Cat#P-8340, Sigma-Aldrich, St. Louis, MO) and NP40 cell lysis buffer. PAPP-A ELISA kits were generously provided by Ansh Labs (Cat#AL-101; Webster, TX), and were used to quantitatively measure human PAPP-A protein expression. This assay does not recognize murine PAPP-A[24 (link)]. Tumors were ranked per PAPP-A expression and split evenly into two cohorts, defining the top 50% as “High PAPP-A” and the bottom 50% as “Low PAPP-A.

Cells were lysed in M‐PER extraction buffer (Thermo Fisher Scientific) in the presence of protease inhibitors (Thermo Fisher Scientific). Five μg (Seps1 and glucose regulated protein (GRP) 94) or 10 μg (GRP78 and heme oxygenase‐1(HO‐1)) of total protein was separated on 4–20% acrylamide gradient gels (NuSep), and transferred to PVDF membranes. After blocking, membranes were incubated overnight with anti‐Seps1 (an in‐house rabbit polyclonal antibody against the COOH terminus of Seps1 generated as described here (Gao et al. 2003, 2004, 2007)), anti‐GRP94 (Cell Signaling, Danvers, USA; #2104), anti‐GRP78 (Cell Signaling; #3183), or anti‐HO‐1 (Assay Designs, Ann Arbor, USA; #SPA‐894) antibodies. This was followed by an incubation with a goat anti‐rabbit HRP‐linked secondary antibody (Cell Signaling; #7074). Bands were detected using ECL chemiluminescence (Invitrogen) and analyzed using Quantity One^® software (BioRad, Gladsville, AUS). The gels were stained with SimplyBlue™ SafeStain (Invitrogen) to validate even loading and protein expression was normalized to the optical density of the total protein per lane.

HaCaT^HRASG12V were plated in culture media with 1% FBS (Invitrogen) overnight before drug treatment. Short-term experiments were treated for 15 minutes before lysis. For time course experiments, media and drug were refreshed at 36 hours. Cells were lysed in MPER Extraction Buffer (Thermo) supplemented with Halt Phosphatase and Protease Inhibitor (Thermo). After clearing lysates with centrifugation, protein concentration was normalized with BCA Assay (Thermo), and 15μg total protein/well was loaded into 1.5mm 10% SDS-Polyacrylamide gels after denaturation in standard SDS-loading dye. Running and transfer were in Tris-Glycine buffer (25 mM Tris, 192 mM glycine). Running buffer was supplemented with 0.1% SDS and transfer buffer with 20% ethanol. Transfer was onto a PVDF Immunobilon Membrane (Millipore).
Primary antibodies for western blotting were from Cell Signaling: rabbit phospho-ERK1/2 (D13.14.4E) and mouse total-ERK1/2 (3A7). Secondary antibodies were from Li-Cor: IRDye 680RD goat anti-rabbit IgG and IRDye 800CW goat anti-mouse IgG. Fluorescent western double staining was developed on the Odyssey fluorescent western system (Li-Cor) graciously supplied by Craig D. Logsdon, PhD.

Blood samples were obtained following approval of the Institutional Review Board (IRB). The IRB also approved the establishment of a serum biorepository at the Scleroderma Center (Protocol Title: “Biochemical and Vascular Alterations in Scleroderma”; IRB Protocol #06F.186). Serum samples were aliquoted and stored at -80°C until assayed. Exosomes were isolated from 0.5 ml of serum by size exclusion chromatography using the SBI (Scientific Bioscience, Palo Alto, CA.) SmartSEC HT EV Isolation System according to the manufacturer’s instructions. Essentially all of the microvesicles isolated employing this procedure are less than 200 nm according to the manufacturer’s brochure. For exosome protein extraction, the isolated exosomes were suspended in 1.0 ml of exosome buffer and proteins were extracted from 0.5 ml of each exosome sample using the M-PER extraction buffer (ThermoFisher Scientific, Watham, MA.) which yielded enough protein to proceed with SOMAscan analysis.

RT-PCR and ELISA were carried out to verify expression of CAR1 and CD16 on lentivirus-transduced or plasmid-transfected cells. Total RNAs were extracted from NK92MI CAR1 dimer, monomer and untransduced cells using TriZol Reagent (Invitrogen). RT-PCR for CAR1, CD16 and beta actin was performed using One-Step RT-PCR kit (New England Biolabs) with corresponding primers (Supplementary Table S1) and was analyzed by agarose gel electrophoresis. RIPA lysis buffer (Santa Cruz) and M-PER Extraction buffer (Thermo Fisher) were used to extract whole cell lysates from NK92MI untransduced and lenti-transduced CAR1 dimer, monomer and GFP only cells. Protease inhibitors (Santa Cruz and Sigma) were added to RIPA (Santa Cruz) and M-PER (Thermo Scientific) lysis buffers, respectively, prior to use. Protein concentrations in the cell lysates were determined by Protein Assay Reagent (Bio-Rad) using Bovine Serum Albumin standard protein (Pierce). CAR1 concentration was assayed by Paired Human Factor VII Antibody Sandwich ELISA (Cedarlane Laboratories) using recombinant L-ICON1 protein as standards following the published procedure with a minor modification (1:200 dilution for capture and detection Abs)^{14 (link)}. CAR1 expression was normalized and presented as ng per μg cell lysate.