Protein extracts from PBMCs were spectrophotometrically quantified with Coomassie Plus Bradford Protein Assay Reagent (Thermo Scientific).
To evaluate the amount of HLA-C, 6 μg of protein was analyzed with MAb L31 (1 μg/ml). Protein lysates from HEK-293T and Chinese hamster ovary (CHO) cells were used as an internal standard and a negative control, respectively.
To evaluate the amount of β2m, 4 μg of protein was immunoblotted with a rabbit anti-β2m antibody (12 μg/ml; Abcam). HEK-293T and HEK-293T β2m-negative (20 (link)) cell lysates were used as an internal standard and a negative control, respectively.
The α/β-tubulin antibody was used as a loading control in accordance with the manufacturer's instructions (Cell Signaling). Horseradish peroxidase-conjugated anti-mouse (Promega) or anti-rabbit (PerkinElmer) IgG was used as a secondary antibody (0.5 μg/ml). The signal was developed with the ECL Advance Western blotting detection kit (Amersham) through the AutoChemi System UVP (BioImaging System).
The amounts of HLA-C and β2m were quantified through densitometric analysis with ImageJ software for OSX. Each sample value was normalized to its α/β-tubulin value. Differences between experiments were normalized with the same internal standard control (HEK-293T cell lysate).
To evaluate the amount of HLA-C, 6 μg of protein was analyzed with MAb L31 (1 μg/ml). Protein lysates from HEK-293T and Chinese hamster ovary (CHO) cells were used as an internal standard and a negative control, respectively.
To evaluate the amount of β2m, 4 μg of protein was immunoblotted with a rabbit anti-β2m antibody (12 μg/ml; Abcam). HEK-293T and HEK-293T β2m-negative (20 (link)) cell lysates were used as an internal standard and a negative control, respectively.
The α/β-tubulin antibody was used as a loading control in accordance with the manufacturer's instructions (Cell Signaling). Horseradish peroxidase-conjugated anti-mouse (Promega) or anti-rabbit (PerkinElmer) IgG was used as a secondary antibody (0.5 μg/ml). The signal was developed with the ECL Advance Western blotting detection kit (Amersham) through the AutoChemi System UVP (BioImaging System).
The amounts of HLA-C and β2m were quantified through densitometric analysis with ImageJ software for OSX. Each sample value was normalized to its α/β-tubulin value. Differences between experiments were normalized with the same internal standard control (HEK-293T cell lysate).
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