Ab227975

We used Immunedeconv [31 (link)] in R along with CIBERSORT [32 (link)] (CIBERSORT, RRID:SCR_016955) to estimate the fraction of immune cells in our transcriptome dataset. CIBERSORT can characterize the heterogeneity of immune cells in bulk RNA-seq. This validated deconvolution approach can differentiate among 22 immune cell types including seven T-cell types, naïve and memory B cells, plasma cells, natural killer (NK) cells and myeloid subsets.
To show that increased LCK expression in IBM muscle compared to controls originates from T cells, we prepared 6 µm frozen sections of four IBM biopsies that were available for further experiments. The slides were immunostained overnight using primary antibodies anti-LCK (RabMab, clone EPR20798-107, Abcam ab227975, RRID: AB_2905531) and anti-CD3 (mouse mAb, clone OKT3, eBioscience™/Thermo Fisher Scientific, RRID:AB_467057). Alexa Fluor 488/594 -conjugated secondary antibodies (Thermo Fisher Scientific) were used for detection, and Hoechst was used for nuclear counterstaining. Microscopic images were obtained using the Zeiss Axio Imager M2 system (Carl Zeiss AG), with 40 × original magnification.

The protein was separated and transferred, and then incubated with 5% milk for 1 h. The protein was incubated at 4°C overnight using antibodies: PSMC4 (ab139184, Abcam), CBX3 (ab213167, Abcam), tropomodulin 3 (TMOD3, ab157215, Abcam), SUZ12 polycomb repressive complex 2 subunit (SUZ12, ab307891, Abcam), LCK proto‐oncogene (LCK, ab227975, Abcam), beta‐transducin repeat containing E3 ubiquitin protein ligase (BTRC, ab71753, Abcam), PI3 kinase p85 (PI3K, 4292S, CST, USA), phospho‐PI3K (BS‐3332R, Bioss, China), Akt (4685S, CST, USA), phospho‐Akt (4060S, CST, USA), mTOR (2983S, CST, USA), phospho‐mTOR (5536S, CST, USA), EGFR (ab52894, Abcam), Bax (ab32503, Abcam), Bcl‐2 (ab182858, Abcam), Caspase 3 (ab32351, Abcam), Cleaved‐Caspase 3 (C‐Caspase, ab32042, Abcam) and GAPDH (1:3000, AP0063, Bioworld). Then secondary antibody was performed and protein was verified using an imaging system (Tanon 5200, China).