Shp 1

Western blotting was performed as described previously using specific antibodies against RORα, actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), pSTAT3, STAT3, JAK1, JAK2, SRC, PIAS3, and SHP-1 (Cell Signaling Technology, Beverly, MA, USA)^{32 (link)}. Quantitative real-time PCR (qPCR) was performed using an ABI StepOnePlus^TM Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using specific primers (Supplementary Table S1). Relative mRNA levels of target genes were estimated using the equation 2^−ΔCt (ΔCt = Ct of target gene minus Ct of β-actin or 18 S rRNA) and are presented relative to the level of the control group, which was designated as 1. The detailed method for qPCR is described in Han et al.^{32 (link)}.

HMC3 cells were seeded into a 6-well plate at 1 × 10⁶ cells/well in EMEM without FBS. After 1-hour incubation with designated treatments, the supernatant was removed, and cells were washed three times by DPBS. The cell lysate was obtained by lysing cells using NP-40 lysis buffer (1% NP40, 50 mM Tris-HCl, pH = 8, 150 mM NaCl) with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (ThermoFisher). After removing debris by centrifugation, the total protein amount normalized by Pierce BCA Protein Assay Kit (ThermoFisher). Protein samples were resolved by 10% SDS-polyacrylamide gels (Biorad) and later transferred onto Immun-Blot PVDF membranes (Biorad). Proteins were probed with specific primary antibodies and secondary antibodies diluted in 5% BSA TBST [21 (link), 24 (link), 29 (link)]. The primary antibodies used are: SYK (1:1000, Cell Signaling 13198S), Phospho-Syk (Tyr525/526) (1:1000, Thermo Fisher MA5-14918), β-Actin (1:1000, Cell Signaling 4970S), SHP1 (1:1000, Cell Signaling 3759S), Phospho-SHP-1 (Tyr564) (1:1000, Cell Signaling 8849S), and LILRB2 (1:1000, Thermo Fisher PA5-46983).
The immunoreactive bands were visualized with the West Pico PLUS Chemiluminescent Substrate (ThermoFisher). The immunoreactive bands were quantified using ImageJ. Three independent treatment replicates were conducted with the representative immunoblot shown.

Mouse macrophages were isolated from BMMCs of 5TGM1/KaLwRij MM mouse models with CD11b MicroBeads (Miltenyi). Mouse macrophages were lysed in the mammalian cell extraction buffer (BioVision) with Protease and Phosphatase Inhibitors (ThermoFisher). Protein lysates were incubated on ice for 10 min and centrifuged at 14,000 × g for 10 min at 4 °C. Proteins were separated with 4–12% Bis-Tris Gel (Invitrogen) at 120 V for 1.5 h. And then transferred to a nitrocellulose (NC) membrane for 1.5 h at 200 mA. The membrane was blocked for 1.5 h with 5% milk at room temperature. Primary antibodies SHP-1 (Cell Signaling Technology, catalog # 3759, clone # C14H6, 1:1000), SHP−2 (Cell Signaling Technology, catalog # 3397, clone # D50F2, 1:1000), Phospho-SHP-1 (Tyr564) (Cell Signaling Technology, catalog # 8849, clone # D11G5, 1:1000), Phospho-SHP-2 (Tyr580) (Cell Signaling Technology, catalog # 5431, clone # D66F10, 1:1000), β-Actin (Cell Signaling Technology, catalog # 4967, 1:1000), were incubated overnight at 4 °C. Anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, catalog # 7074, 1:1000) were incubated for 2 h. ECL Substrates (Bio-Rad, catalog # 1705060) were used for exposure. Western Blotting imaging was exposed with a Bio-Rad ChemiDoc XRS⁺. The images were analyzed by Image Lab Software (Bio-Rad).