Cocktail set 1

Excised hind paws were flash frozen in liquid nitrogen and homogenized in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) supplemented with Protease Inhibitor (Cocktail Set I; EMD Millipore, Billerica, MA) using a Tissue Tearor (BioSpec; Model 985370). Homogenates were incubated on ice for 20 minutes, centrifuged at 2000 rpm for 10 minutes at 4°C, and lysates stored at −80°C. Histamine (Neogen Corporation, Lansing, MI), and cytokines IL-1β, IL-6, and IL-10 (R&D Systems, Minneapolis, MN) were quantified using ELISA according to manufacturers’ instructions. Cytokine concentrations were normalized to the total protein concentration in each sample using the DC Protein Assay Kit I (Bio-Rad, Hercules, CA). All absorbances were recorded using a PowerWave XS microplate spectrophotometer (BioTek Instruments, Winooski, VT).

Protein extraction was performed with a lysis buffer containing Tris 50 mM pH 7.4, NaCl 150 mM, SDS 0.1%, Triton X-100 1% (Sigma), Nonidet P-40 1% (Igepal), proteinase (Cocktail Set I, Merck; Pefabloc, Roche), and phosphatase (Phostop, Roche) inhibitors. Equal protein amounts were separated with precast gels, transferred to a polyvinylidene difluoride membrane as described [23 (link)], blocked, and incubated overnight with primary antibodies against phosphorylated SMAD3 (pSMAD3) (#07-1389, Merck Millipore) and α-Tubulin (#2144, Cell Signaling Technology; used as loading control) as well as against P4HA2 (#13758-1-AP, ProteinTech), α-SMA (#A5228, Sigma-Aldrich), Akt^pS473 (#4060S), Akt (#9272S, Cell Signaling) and β-Actin (#A1978, Sigma-Aldrich; used as loading control). Protein bands were labeled and visualized by chemiluminescence (Chemidoc Imaging Systems, Bio-Rad). Band intensities were analyzed with ImageJ and normalized to the corresponding loading control. Since pAkt and Akt have the same molecular weight (60kDa), samples were loaded in two independent precast gels and ran in parallel; α-Tubulin levels were assessed as a loading control (referred to as α-Tubulin⁽¹⁾ in the “pAkt membrane” and α-Tubulin⁽²⁾ in the “total Akt membrane”). To obtain the final pAkt/Akt ratio, the pAkt/α-Tubulin⁽¹⁾ densitometry ratio was divided with the corresponding Akt/α-Tubulin⁽²⁾ ratio.