Tris glycine mini protein gel

Protocols for plasma membrane preparation, SDS/PAGE, and electrotransfer on to polyvinylidene difluoride membranes (Pierce, Rockford, IL) were identical with those previously described [20 (link),39 (link)]. For fractionation of crude membrane fractions, 10% SDS/PAGE gels were used. For fractionation of biotinylated surface membrane proteins, Novex™ 4–20% Tris/glycine mini-protein gels were used. Anti-HA antibody (Covance; Princeton, NJ), anti-Myc antibody (Covance), and IRDye800-conjugated secondary antibody were used with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) to identify HA- and Myc-tagged hPCFT proteins. Anti-β-actin (Sigma) or anti-Na⁺/K⁺-ATPase (Novus Biologicals, Littleton, CO) antibodies were used to establish equal protein loading. Densitometry values were calculated using the Odyssey Infrared Imaging System and software package.

Cells were harvested in RIPA buffer (150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH7.5, and 2 mM EDTA) with 1% protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein were separated by SDS/PAGE on Novex 4–20% Tris-Glycine Mini Protein Gels (1.0 mm, 10 well) and analyzed by immunoblotting. Western blotting was prepared by standard procedures using rabbit anti-COX2 (Abcam, ab15191), rabbit anti-TSG6 (Abcam, ab128266), rabbit anti-IL1RA (Abcam, ab124962), rabbit anti-heme oxygenase 1 (Abcam, ab13243), and beta-actin (Santa Cruz, sc-47778) antibodies. Gel images were scanned and the density of the protein bands was quantified as a ratio to the loading control by using MCID Analysis 7.1 software (Imaging Research).

Q5^® Site-Directed Mutagenesis Kit was ordered from New England Biolabs (Ipswich, MA). All oligonucleotides were synthesized by Eurofins MWG Operon (Huntsville, AL). Chinese Hamster Ovary-suspension (CHO-S) cells, FreeStyle^™ CHO Expression Medium, Fetal Bovine Serum (FBS), 4-12% Tris-Glycine Mini Protein Gel, and SimpleBlue SafeStain were obtained from Invitrogen (Grand Island, NY). TransIT-PRO^® Transfection Kit was purchased from Mirus (Madison, WI). The rmp Protein A Sepharose Fast Flow was from GE Healthcare Life Sciences (Pittsburgh, PA). (–)-Cocaine was provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program (Bethesda, MD); and [³H](–)-Cocaine (50 Ci/mmol) was obtained from PerkinElmer (Waltham, Massachusetts). All other materials were from Sigma-Aldrich (St Louis, MO) or Thermo Fisher Scientific (Waltham, MA).