Bam8 22

Histamine dihydrochoride (Sigma-Aldrich; 10 μg/10μl), BAM8-22 (Tocris Bioscience; 10 μg/10μl), and β-alanine (Sigma Aldrich; 90 μg/10μl) were each prepared in sterile extracellular fluid ^{29 (link)}. Intradermal injection of a given chemical was administered in the ACD or control site 24–72 hours post-challenge. Subjects reported the intensity of any spontaneous itch, pricking/stinging or burning they were experiencing from the ACD and control sites prior to injection. They were instructed to disregard the initial prick of the needle entering the skin, and to report the highest magnitude of each sensation they felt every 30 seconds by moving a cursor along the gLMS on a computer screen ^{28 (link), 29 (link)}. Numerical values (not presented to the subject) of 1 to 100 were derived from the cursor position. In each testing period, only one injection was given in each site. The three chemicals were administered in no specific order over 24–72 hours post-challenge.

BAM8-22 was obtained from Tocris Bioscience (Ellisville, MO, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). SADBE was dissolved in a vehicle of acetone. Histamine dihydrochloride, BAM8-22 and bradykinin were each dissolved in a vehicle of sterile, normal saline. The doses of all chemicals used in this study were based on the results of pilot studies or published findings [3 (link),4 (link),16 (link)].

Stock solutions were freshly prepared as instructed by the manufacturer. BAM8-22 and JHU58 were diluted in saline or extracellular solution. JHU58 was synthesized at the Johns Hopkins University. BAM8-22 was purchased from Tocris Bioscience (Bristol, UK). TAK-243 was obtained from Chemietek (Indianapolis, IN, USA). Other drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used: anti-Myc (Cell Signaling Technology, Cat# 2276, RRID: AB_331783, Cat# 2278, RRID: AB_490778), anti-HA (Cell Signaling Technology, Cat# 3724, RRID: AB_1549585), anti-ubiquitin (Cell Signaling Technology, Cat# 43124, RRID: AB_2799235), anti-GLUT1 (Cell Signaling Technology, Cat# 12939, RRID: AB_2687899), anti-Rab11a (Cell Signaling Technology, Cat# 5589, RRID: AB_10693925), anti-actin (Millipore, Cat# MAB1501, RRID: AB_2223041), and anti-GAPDH (Millipore, Cat# ABS16, RRID: AB_10806772).