Ab2872

Eighty pairs of RCC tissues (RCTs) and their counterparts, autologous para-cancerous kidney tissues (PKTs), were cut into 5-µm thickness sections for hematoxylineosin (HE) and immunohistochemistry (IHC) analysis according to our previous protocols [28 (link)]. The anti-ATP1A1 antibody (1:400, ab2872, Abcam, UK) was used to detect ATP1A1 level among RCTs and PKTs. A biotinylated anti-goat IgG (ZB-2306, ZSGB-BIO Corp., Beijing, China) was used as a secondary antibody. Then reaction with 3,3′-diaminobenzidine substrate solution (Dako Cytomation GmbH) and counterstaining with hematoxylin were performed. Five independent fields at ×50 magnification for positive cells were chosen to evaluate the intensity and percentage. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The staining percentage was scored as 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The immunoreactivity score for each tissue sample, ranging from 0 to 12, was measured as immunostaining intensity multiplied by percentage of positive cells [24 (link), 25 ]. The expression level of ATP1A1 was defined as our previous work [29 (link)]. The score of 0 was defined as negative, scores of 1–4 as low expression, and scores over 4 as high expression. The final score was an average value calculated from evaluation scores by two pathologists separately.

OS-RC-2 and 786-0 cells were transfected with pYR-ATP1A1 or pYR plasmids for 48 h, and cell pellets were collected. Cell pellets and grated tissues were dissolved with RIPA buffer (Cat.# P0013C, Beyotime, China) to extract proteins. 100 μg proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Cat.# IPVH00010, China). After blocking with 5% skim milk (OXOID), the PVDF membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies against ATP1A1 (1:200, ab2872, Abcam, UK), MEK1/2 (1:500, sc436, Santa Cruz, USA), p-MEK1/2(1:500, sc-81503, Santa Cruz, USA), ERK (1:500, sc-292838, Santa Cruz), p-ERK (1:500, sc-136521, Santa Cruz) β-actin (1:500, sc-1616, Santa Cruz, USA), and GAPDH (1:1000, sc-365062, Santa Cruz) were respectively diluted in TBST buffer (50 mM Tris–HCl, with 150 mM NaCl, 0.1% Tween-20, pH 7.4). Then the PVDF was incubated with the HRP-tagging secondary antibodies (ZSGB-BIO, China) at 37 °C for 1 h. Signals were finally detected with Western blot reagent ECL (Amersham Biosciences).