Ab190114

The transfected cells were fixed in 4% paraformaldehyde lasting 30 min. And, 0.5% Triton X-100 was applied to cover the slides and incubated at room temperature for 10 min. The sample was blocked with 10% goat serum for 1 h, and then washed thrice with 1% serum blocking solution. The primary antibody against MCU (1:100; sc-515,930; Santa Cruz, USA), MICU1 (1:1000; ab190114; Abcam, USA) and MICU2 (1:1000; ab101465; Abcam, USA) was added dropwise to the slices. After washing six times with 1% serum blocking solution, the sections were incubated with secondary antibodies Alexa Fluor® 488 Conjugate (1:100; ZF-0512; ZSGB-BIO, China) and Alexa Fluor® 594 Conjugate (1:100; ZF-0513; ZSGB-BIO, China) at room temperature for 2 h. Then, the sections were incubated by DAPI at room temperature for 5 min in the dark. The images were captured using an immunofluorescence microscope. The Image-Pro Plus 6.0 image processing system was used to measure the average optical density of the protein.

Paraffin-embedded tissue sections were incubated with primary antibodies labeled with fluorescent substance against MCU (1 : 100; sc-515930; Santa Cruz, US), MICU1 (1 : 1000; Abcam, USA; ab190114), MICU2 (1 : 1000; Abcam, USA; ab101465), and Nrf2 (1 : 1000; Proteintech, China; 16396-1-AP) overnight at 4°C, followed by secondary antibodies Alexa Fluor® 488 conjugate (1/100; ZSGB-BIO, China; ZF-0311, 1/100; ZSGB-BIO, China; ZF-0514) and Alexa Fluor® 594 conjugate (#ZF-0513, 1 : 100, ZSGB-BIO, China) at room temperature for 2 h. The images were observed under an immunofluorescence microscope.

The protein was extracted from tissues or cells. The BCA protein quantification kit was employed for quantitative detection of protein sample. The protein sample was added with 5× loading buffer, mixed, and boiled at 100°C for 5 min. Each loading hole was filled with 40 μg protein sample. With 10% separating gel and 5% concentrated gel, electrophoresis was presented under the condition of 90 V constant pressure. The polyvinylidene fluoride (PVDF) membrane was soaked in the transfer buffer and protein was transferred to the membrane in an ice bath at 200 mA for 2 h. The membrane was placed in 5% bovine serum albumin and incubated at 37°C for 2 h. The membrane was incubated with primary antibodies against MCU (1:1000; sc-515,930; Santa Cruz, USA), MICU1 (1:1000; ab190114; Abcam, USA), MICU2 (1:1000; ab101465; Abcam, USA), N-cadherin (1:100; ab98952; Abcam, USA), TGF-β (1:1000; ab215715; Abcam, USA), vimentin (1:1000; 10,366-1-AP; Proteintech, China), Smad4 (1:1000; ab230815; Abcam, USA) and β-actin (1:2000; 20,536-1-AP, Proteintech, China) at 4°C overnight. After taking out the PVDF membrane, it was incubated with IgG secondary antibody (1:5000; ZB-2305; ZSGB-BIO, China) at room temperature for 2 h. After electrochemiluminescence, the image was captured for quantitative analysis with ImageJ.