Gapdh loading control monoclonal antibody ga1r

ARS-1620 was a free sample from ChemieTek (Indianapolis, IN). The monoclonal antibodies against ABCB1 (clone F4, Cat # SAB4200775) and other chemicals were purchased from Sigma Chemical Co (St. Louis, MO) except otherwise specified. Antibiotics (penicillin/streptomycin), trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Hyclone (Waltham, MA, United States). The Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG secondary antibody (Cat # 7076S, lot #: 32) was the product of Cell Signaling Technology Inc. (Danvers, MA, United States). The GAPDH loading control monoclonal antibody (GA1R) (Cat # MA5-15738, lot #: SA247966), Alexa Fluor 488 conjugated goat anti-mouse IgG cross-adsorbed secondary antibody (Cat # A32723) were acquired from Thermo Fisher Scientific Inc. (Rockford, IL, United States). [³H]-paclitaxel (15 Ci/mmol) was obtained from Moravek Biochemicals, Inc. (Brea, CA).

The lysates were cleared by centrifugation (13 000×g, 13 min) and equalized for the total protein amount (DC Protein Assay, BioRad). Agarose-bound GST Rhotekin (20 μg) was added into each lysate and rotated in 4°C for 40 min. After incubation, the beads were washed three times in lysis buffer and resuspended in SDS-PAGE sample buffer. The samples were resolved using SDS-PAGE and analyzed by immunoblotting with respective antibodies: RhoA (67B9) mAb (2117, Cell Signaling Technology) and GAPDH Loading Control Monoclonal Antibody (GA1R, MA5-15738, Invitrogen).

Immunoblotting was performed according to Invitrogen protocols for Mini Gel Tank and iBlot2 dry system. In brief, cell lysates were prepared to incubate cells with RIPA buffer (Invitrogen) containing Halt Protease Inhibitor Cocktail (Invitrogen) and quantified using BCA assay (Thermo Fisher Scientific). EVs lysates were prepared incubating cells with 50 μl of RIPA buffer (Invitrogen) containing Halt Protease Inhibitor Cocktail (Invitrogen) and sonicated for 10 min. In all, 25 μl of EVs lysate was loaded on the gel. Proteins were separated using NuPAGE Bis-Tris 4–12% gels (Invitrogen) and transferred to nitrocellulose membranes using the iBlot2 dry method. Membraned were blocked for 40 min in 4% milk and incubated with the primary antibodies in 4% milk 1.5 h at room temperature for anti-CHMP2A antibody or ON at 4 °C for all the other antibodies used. After incubating the membrane with the appropriate secondary antibody conjugated to horseradish peroxidase, protein levels were detected using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore). CHMP2A overexpression lysate (OL) control used in Fig. 2c was purchased from Origene. CHMP2A Antibody, rabbit polyclonal, Proteintech, cat. no. 10477-1-AP (1:600). GAPDH Loading Control Monoclonal Antibody (GA1R) (1:1000), Invitrogen, cat. no. MA5-15738. CD9, clone (D8O1A) Rabbit mAb, Cell Signaling, cat. no. 13174S (1:1000).