Rabbit anti fbxo28 antibody

Total protein was extracted by using RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China) after being lysed on ice, ultrasonicated and centrifuged. Based on the protein concentration determined by the BCA protein detection kit (Beyotime, China), an equally concentrated protein mixture was prepared with loading buffer solution (Biosharp, Hefei, China). The target proteins were separated via 8% or 10% SDS‒PAGE gel. Then proteins were subsequently transferred to a PVDF membrane at 300 mA for 90 min to complete the transmembrane. After 5% skim milk powder was added for 1 h, the membrane was incubated with primary antibodies at 4 ℃ overnight, which include rabbit anti-FBXO28 antibody (1:1000; abcam), rabbit anti-TGF-b1antibody (1:1000; abways), rabbit anti-Smad2/3 antibody (1:1000; CST), rabbit anti-p-Smad2/3 antibody (1:1000; CST), rabbit anti-N-cadherin antibody (1:1000; Proteintech), rabbit anti-E-cadherin antibody (1:1000; Proteintech), mouse anti-GAPDH antibody (1:5000; Proteintech) and rabbit anti-β-Tubulin (1:2000; Proteintech). After the corresponding secondary antibody was added and incubated at room temperature for 1 h, the proteins were visualized via an enhanced chemiluminescence kit (Meilunbio, China).

The paraffin sections containing tissues were placed in a drying oven at 60 ℃ for 2 h to finish baking the slices, which were subsequently dewaxed with xylene and gradient ethanol. The tissues were soaked in sodium citrate buffer solution with a heated boil, after which antigen retrieval was completed. The endogenous enzyme activity was inactivated by hydrooxidase, 5% bovine serum albumin (BSA) was used to block the tissue sections, which were incubated overnight at 4 ℃ with primary antibodies, including rabbit anti-FBXO28 antibody (1:100; Abcam), rabbit anti-N-cadherin antibody (1:200; Proteintech), rabbit anti-E-cadherin antibody (1:100; Proteintech) and mouse anti-Ki67 antibody (Zsbio, China). After the sections were incubated at room temperature with the corresponding secondary antibodies, DAB and hematoxylin were used to stain the sections. The staining intensity was scored according to the following criteria: 0 (blue, feminine), 1 (light yellow, weakly positive), 2 (brownish, yellow, moderately positive), or 3 (dark brown, strong positive). The staining area was scored as 1 (≤ 25% of positive cells), 2 (26–50% of positive cells), 3 (51–75% of positive cells), or 4 (≥ 75% of positive cells). The IHC staining was calculated by multiplying the staining intensity score and positive staining percentage score [27 (link)].