Tsbp1

Tissue sample (100 µg) was added to the RIPA buffer (Thermo, Waltham, MA, USA) for homogenization and then subjected to centrifugation to obtain the supernatant. Protein concentration was quantified using the BCA assay kit (Thermo, Waltham, Massachusetts). The tissue lysates were separated using 10% SDS–PAGE gel for 90 min and transferred to polyvinylidenefluoride membranes (Roche, Basel, Switzerland) for 2 h. After blocking with 5% BSA in Tris-buffered saline for 1 h. Then, the blots were incubated with primary antibodies for FoxA1 (Cell signaling, Beverly, MA, USA), HMGN1 (Cell signaling, Beverly, MA, USA), HMGN2 (Cell signaling, Beverly, MA, USA), HMGN3 (Abcam, Cambridge, UK), and TSBP1 (Abcam, Cambridge, UK) overnight, after which they were reacted with an HRP-conjugated secondary antibody for 2 h. An enhanced chemiluminescence prime western blotting detection reagent (GE Healthcare, Chalfont St. Giles, UK) was used to visualize the protein bands. The density of respective bands was analyzed with the iBright 1500 (Thermo, Waltham, MA, USA).

One µg of tissue lysate sample was mixed with 50 µL of coating buffer (0.2 M Sodium bicarbonate pH 9.4) and incubated overnight at 4 °C in a 96-well plate (Corning, NY, USA). Next, it was washed three times with wash buffer (TBS containing 0.05% Tween 20, pH 7.2) and then incubated for 7 h at room temperature in the blocking buffer (3% BSA in TBS-T). The primary antibodies against FOXA1 (cell signaling), HMGN1 (cell signaling), HMGN2 (cell signaling), HMGN3 (Abcam), and TSBP1 (Abcam) were added to each well, followed by overnight incubation at 4 °C. After washing plate three times with TBS, HRP-conjugated rabbit antibody was added to each well and incubated for 3 h. Finally, after washing three times with TBS, and the TMB substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well. After 5–25 min, when the color changed appositely, a stop solution was added to each well. The absorbance of the plate was measured using a microplate reader (TECAN, Switzerland) at 450 nm.