Gtx213110

Human ORS and DP cells or skin tissue were lysed (RIPA lysis buffer; #20‐188; Merck Millipore). The obtained protein was then separated by 8% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Amersham). The protein transferred membranes were incubated overnight with the specific antibodies. The following antibodies were used: β‐actin (#MA5‐15739, 1:5,000; Thermo Fisher), phospho‐ERK1/2 (#9101, 1:1,000; Cell Signaling), total ERK1/2 (#9102, 1:1,000; Cell Signaling), phospho‐AMPK (#2535, 1:1,000; Cell Signaling), and total AMPK (#2532, 1:1,000; Cell Signaling). Then, the membranes were washed and incubated with anti‐rabbit IgG or anti‐mouse IgG antibodies (horseradish peroxidase‐conjugated, GTX213110, GTX213111, 1:10,000; GeneTex) at 25°C for 1 h. Antibody‐antigen complexes detected by enhanced chemiluminescent substrate (Thermo Fisher) were captured and quantified by Amersham imager 680 systems (GE Healthcare).

Oxaliplatin was purchased from Sanofi Pharmaceutical Company (NY, USA). The procedure for synthesizing the CTSS inhibitor RJW-58 was described previously 14 (link). The following primary antibodies were purchased: Cathepsin S (Thermo Fisher catalog: #PA5-81369), EBF (Santa Cruz, #sc-137065), ATF-3 (Biossua, #BS-0519R), α-tubulin (Sigma, MO, USA), IRF-1 (Bio-Rad, #VPA00801), CBP (GeneTex, #GTX101249), Iba1 (Abcam, #ab-5076), NeuN (Novus, #NBP2-10491), IL-10 (Abcam, #ab189392), STIM1 (Cell signaling (D88E10), #5668S), CD11b (Abcam, #ab52478), CD45 (BD Biosciences, #553079), STAT3 (BD, #50402), P-STAT3 (Cell signaling, #9145S), NFκB (Cell signaling, #4727S), OR5B3 (Abcam, #ab186624), OR5M3 (LSBio, #LS-C200406), and β-actin (GeneTex, #GTX109639). Horseradish-peroxidase-conjugated secondary antibodies were purchased from GeneTex (#GTX213110, #GTX213111, #GTX224125, and #GTX232040).

Brain tissues were processed for immunofluorescent detection. The primary antibodies used were anti-ionized calcium-binding adaptor molecule-1 (Iba-1; 1:250; GTX100042, GeneTex, Alton Pkwy Irvine, CA, USA) and anti-MAP2 (1:200; GTX11267, GeneTex, Alton Pkwy Irvine, CA, USA). The brain sections were incubated with primary antibodies overnight, then washed and incubated with either Alexa Fluro 488- (1:500; GTX213111, GeneTex, Alton Pkwy Irvine, CA, USA) or Alexa Fluro 594- (1:500; GTX213110, GeneTex, Alton Pkwy Irvine, CA, USA) tagged secondary antibodies at room temperature. The number of cells positive for Iba-1 and MAP2 was counted in three non-overlapping fields in the cortex and hippocampus at ×200 magnification. Next, the sections were incubated in a 1:200 dilution of a TRITC-conjugated goat anti-rabbit secondary antibody and FITC-conjugated goat anti-mouse secondary antibody for 1 h at room temperature and then washed three times with PBS for 10 min. Then, the sections were stained with DAPI solution for 10 min at room temperature. Finally, fluorescence images were obtained by fluorescence microscopy (Leica DM 6000B; Mannheim, Germany), and the number of cells was counted with the ImageJ software.