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The analyte molecules were separated on Phenomenex kinetex C18 column 100 (2.6 m, 50 3 mm) with the mobile phase composed of Water/0.1% formic acid (A) and Acetonitrile (B) at the flow rate of 0.5 ml/min with the column temperature maintained at 40 C and the sample of injection was 5 l

48 h after transfection, cells were washed twice with pre-warmed PBS and fixed with 4% paraformaldehyde (PFA) for 1015 min

Optimize first: Gel percentage, transfer time, membrane choice, sample denaturation, and reducing conditions

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