Fab3518a

To construct the LEMP-HA, Flag-LEMP plasmids, the corresponding sequence was HA or Flag tagged and inserted into the pHAGE-fEF1a-IRES-ZsGreen vector. LEMPΔATG-HA plasmid was obtained using mutagenesis. To construct LEMP-Flag-px330, a single-guide RNA (sgRNA) specific to the C-terminal coding sequence of mLEMP locus was cloned into the px330. The donor plasmid was made with a Flag tag in-frame with the LEMP coding sequence and flanked by ~500 base pair homology arms specific to the LEMP locus. The zebrafish cDNA of LEMP gene was amplified and cloned into pCS2+ vector. To construct the plasmid used for synthesis of antisense RNA probe, the relevant sequence of LEMP was cloned into pCS2+ vector. To make the construct of LAT-EGFP, the zLEMP-AMO targeting sequence was inserted into the corresponding start codon region of EGFP in pCS2+ vector.
The antibodies against Flag (Sigma, F3165), HA (Sigma, H6908), GAPDH (Abcam, ab8245), MHC (Upstate, 05-715), Mitotracker (Invitrogen, M7510), Gm130 (BD, 610823), Calnexin (Santa Cruz, SC-6465), LEMP (ABclonal, A18310), CD31 (BD, 562861), CD45 (eBioScience, 45-0451-82), CD11B (eBioScience, 45-0112-82), Sca1 (eBioScience, 56-5981-82), CD34 (BD, 553733), and integrin-α-APC (R&D, FAB3518A) were purchased.

MACS-isolated MuSCs cultured for 4–5 days were detached using 0.25% trypsin/EDTA, followed by centrifugation and resuspended in 2% FBS/PBS. The cells were incubated with APC-conjugated-a7-integrin antibody (FAB3518A, R&D Systems) for 30 min on ice, followed by the addition of propidium iodide (PI) at a 1:500 (v/v) ratio. The cells were either analyzed using a flow cytometer (CytoFLEX S; Beckman Coulter, Brea, CA, USA) or sorted using a MoFlo XDP flow cytometer (Beckman Coulter). Debris and dead cells were excluded by forward scatter, side scatter, and propidium iodide (PI) gating. Gates were defined based on isotype or WT control fluorescence. Data were analyzed using FlowJo software ver. 10.7.1 (BD Biosciences, NJ, USA).