Ab123946

Mouse spinal cord tissues (epicenter ± 0.3 cm) and DRG samples were collected and homogenized with 10 strokes of a homogenizer at 4°C in protein extraction buffer. Subsequently, protein concentrations were determined by the BCA method, and equal amounts of proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk or 5% Bovine Serum Albumin (BSA) for 2h at room temperature, the membranes were incubated overnight at 4°C with the following corresponding primary antibodies: anti-PD-L1 (1:1000, ab213480, abcam), anti-TRPV1 (1:1000, 508564, ZEN BIO, Chengdu, China), anti-iNOS (1:1000, ab178945, abcam), anti-Arg1 (1:1000, 16001-1-AP, proteintech, Wuhan, China), anti-β-actin (1:1000, ab123946, abcam), anti-ERK (1:1000, 4695, CST, Massachusetts, USA), anti-pERK1/2 (1:1000, 4370, CST), anti-p38 (1:1000, 8690, CST), anti-pP38 (1:1000, 4511, CST), anti-JNK (1:1000, 9252, CST), anti-pJNK (1:1000, 9255, CST). After washing and incubating with species-matched secondary antibodies (1:5000) at RT for 2 h, immunoreactive bands were visualized using ECL reagents (PE0010, Solarbio, Beijing, China) and the density of protein was quantified by the ImageJ (National Institutes of Health, Bethesda, MD, USA)

The plasmids for SOX2 shRNA and mammalian and lentivirus-mediated protein overexpression were previously reported (Herreros-Villanueva et al., 2013 (link)). The plasmids for FGFR knockdown were constructed using pLKO-1 lentiviral expression vector and the detailed gRNA sequences are listed in Table 1. HA-tagged wild type AKT (HA-AKT), its dominant negative mutant version (K179M, AKT-KD) and HA-ubiquitin (HA-Ub) expression plasmids were acquired from Addgene. All chemical reagents and antibodies used in this study are commercially available. FGFR and AKT inhibitors were purchased from MCE (MedChemExpress), LLL12 was purchased from KareBay Biochem. The following primary antibodies were purchased from Cell Signaling Technology: P-AKT (CST, 4060#), AKT (CST, 4685#), P-STAT3 (CST, 9145#) STAT3 (CST, 9139#), P-FGFR (CST, 3476#), FGFR1 (CST, 9740#), FGFR2 (CST, 23328#), SOX2 (CST, 3579#/4900#), CD44 (CST, 3570#), α-Tubulin (CST, 3873#), GAPDH (CST, 2118#), Histon-H3 (CST, 4499#), FLAG (CST, 14793#), HA (CST, 3724#). Antibody against CD24 was from Abcam (ab123946).

Total cell lysates were resolved on sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto polyvinylidene fluoride membranes. After blocking in 5% (w/v) dry milk in Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with anti-CD68 (ab125212, Abcam, Cambridge, UK), anti-CD163 (ab182422, Abcam), anti-CD204 (ab123946, Abcam), anti-CD64 (PA5-24855, Thermo Fisher Scientific), anti-CD16 (MA1-19006, Thermo Fisher Scientific), or anti-β-actin (ab8227, Abcam) primary antibody at room temperature for one hour. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (ab205718, Abcam). Signals were detected using a BioSpectrum Imaging System (UVP LLC, Upland, CA, USA).