Alexa 488 conjugated secondary antibody

Cells were fixed in paraformaldehyde (Solarbio, Beijing, China) and permeabilized in 0.5% Triton X-100. For Fluorescence in situ hybridization (FISH) assay, Cy3-labeled probes of LINC00963 were utilized to examine the expression in Ishikawa cells and ECSCs. Hybridization was performed with a Fluorescent in Situ Hybridization Kit (RIBO Bio, Guangzhou, PR China) for 12 h away from light. For Immunofluorescence (IF) assay, Ishikawa cells and ECSCs were treated with antibodies against UPF1 (1:500, Abcam, Cambridge, UK) for 2 h then labeled with Alexa 488-conjugated secondary antibody (1:400) for 1 h. The nuclei of Ishikawa cells and ECSCs were subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China). The fluorescence images were captured by the confocal laser microscope (Olympus Optical, Tokyo, Japan).

HEK293 cells transfected with wild‐type GPR21 and GPR21 mutants were harvested and incubated with phosphate buffer solution (PBS) plus 0.5% bovine serum albumin (BSA) for 15 min, followed by incubation with anti‐FLAG antibody (Thermo Fisher) at 4°C for 1 h. After washing with PBS three times, the cells were incubated with Alexa‐488‐conjugated secondary antibody (Beyotime) at 4°C for 1 h. After washing with PBS three times, the cells were subjected to flow cytometric analysis on an AccuriTM C6 Plus flow cytometer (BD). FL1 channel was employed to record fluorescent signal derived from Alexa‐488. The surface expression levels of GPR21 mutants were normalized to that of wild‐type GPR21 (100% level).

Monocyte chemoattractant protein-1 (MCP-1) was purchased from R&D Systems (MN, USA). Primary antibodies for human RAGE, IkBα, NF-κB p65, phosphorylated NF-κB p65, CCR2, and GAPDH were purchased from Abnova (Taipei, Taiwan). Alexa 488 conjugated secondary antibody, Alexa Fluor 555 Phalloidin, and DAPI were purchased from Beyotime (Shanghai, China). The siRNA duplexes against human RAGE (ID: 110859) were purchased from Ambion Life Technologies (NY, USA). The RAGE shRNA lentiviral particles and control shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (USA). The CCR2 neutralizing antibody and CCR2 Isotype control were purchased from Santa Cruz Biotechnology (USA). The CCR2 antagonist (C₂₈H₃₄F₃N₅O₄S, CAS 445479-97-0) was purchased from Merck Millipore (USA).