Glyceraldehyde 3 phosphate dehydrogenase gapdh

For assessing mutant EPRS association with MSC scaffold proteins, 1.5 × 10⁶ of HEK293T shEPRS-inducible stable cells were seeded on 10 cm dishes and shRNA expression was induced with doxycycline the following day. Cells were transfected with 10 μg of pcDNA3 EPRS-FLAG (WT or mutant), using the PEI method (63 (link)) 24 h post doxycycline treatment. Cells were harvested 48 h post transfection and lysed in cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Sigma-Aldrich).
For immunoprecipitation studies, 5 μg FLAG M2 mouse monoclonal antibody (Sigma-Aldrich F1804) or mouse IgG isotype control (Invitrogen 10400C) was conjugated to 25 μl Dynabeads protein G (Invitrogen) by incubating in 200 μl phosphate buffered saline with 0.01% Tween-20 (0.01% PBS-T) at room temperature for 10 min. Immunoprecipitation was performed by incubating 250 μl lysates (∼1200 μg protein) with antibody-conjugated beads overnight at 4 °C. Beads were washed three times with 0.02% PBS-T and boiled in SDS-PAGE loading buffer, followed by immunoblotting with the following antibodies: EPRS (Novus Biologicals NBP1-84929), AIMP2 (Thermo Scientific PA5-31306), AIMP3 (Invitrogen PA5-28283), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bio-Rad). Immunoblots were developed for chemiluminescence and were imaged on an Amersham Imager 680 (Cytiva).

After 48 h of transfection, total RNA was extracted from transfected cells using TRIzol RNA isolation reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. A universal real-time PCR kit (Solarbio, Beijing, China) was used to reverse transcribe the isolated RNA into complementary DNA (cDNA). A fluorescence quantitative PCR instrument (CFX96 Touch; Bio-Rad) was used to measure gene expression. Real-time PCR was performed according to the manufacturer’s instructions. All procedures were performed on ice. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (Bio-Rad, Hercules, CA, USA) was used as an internal reference gene, and each sample was processed in triplicate. Relative expression quantitative (RQ) values of different specimens were calculated using the 2− ΔΔCt method [13 (link)]. The primers used for real-time PCR are listed in Table 1.Table 1.

Primers used sequences for real-time PCR

Gene	Species	Forward (5’→3’)	Reverse (5’→3’)
PD-L1	Human	GCTGCACT AATTGTCTATTGGGA	AATTCGCTTGTAGTCGGCACC
LINC00244	Human	TTCCTCCATTTTAAAATTTTATTC	ATACAATGTAAGGATTTCCATAG
GADPH	Human	ACCCACTCCTCCACCTTTGAC	TGTTGCTGTAGCCA AATTCGTT
U6	Human	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
18S	Human	GGCGCCCCCTCGATGCTCTTAG	GCTCGGGCCTGCTTTGAACACTCT