Tissues or cells were lysed using 100 μL RIPA lysate supplemented with protease inhibitor cocktail on the ice for 30 min, which were then centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was harvested and stored at −80 °C. BCA method was used to measure the concentration of total protein. Protein samples were transferred onto the PVDF membrane (Millipore, USA) and blocked by 5% skimmed milk powder for 2 h. The membranes were incubated with primary antibodies including anti-E-cadherin (1:1000; ab76055; Abcam, USA), anti-Vimentin (1:2000; ab92547; Abcam, USA), anti-N-cadherin (1:1000; ab18203; Abcam, USA), anti-HIF-1α (1:1000; 20,960-1-AP; Proteintech, China), anti-MCU (1:400; sc-515,930; Santa Cruz, USA), anti-VEGF (1:1000; ab52917; Abcam, USA), anti-TGF-β (1:1000; ab92486; Abcam, USA), anti-ITGB1 (1:1000; 12,594-1-AP; Proteintech, China), anti-MMP-2 (1:100; 10,373-2-AP; Proteintech, China) and anti-β-actin (1:1000; 20,536-1-AP; Proteintech, China) for 4 °C overnight. After that, the membranes were incubated with horseradish peroxidase IgG antibodies for 30 min at room temperature. Protein blots were visualized using Chemiluminescence kit (ECL).
Sc 515 930
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-515,930 is a laboratory product offered by Santa Cruz Biotechnology. It is a reagent used in scientific research applications. The core function of Sc-515,930 is to facilitate specific experimental procedures, though its detailed description and intended use are not available in this factual, unbiased response.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugate, by ZSGB-BIO (3 mentions)
Ab190114, by Abcam (3 mentions)
Alexa fluor 594 conjugate, by ZSGB-BIO (3 mentions)
Ab101465, by Abcam (2 mentions)
Ab76055, by Abcam (1 mentions)
Ab52917, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab92486, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Ab215715, by Abcam (1 mentions)
Ab98952, by Abcam (1 mentions)
Zb 2305, by ZSGB-BIO (1 mentions)
5 protocols using sc 515 930
1
Western Blot Analysis of EMT Markers
2
Immunofluorescence Analysis of Mitochondrial Proteins
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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.
3
Immunofluorescence analysis of MCU and CD34
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Paraffin-embedded tissue sections were incubated with primary antibodies labeled with fluorescent substance including anti-MCU (1:100; sc-515,930; Santa Cruz, USA) and anti-CD34 (1:100; ab81289; Abcam, USA) overnight at 4 °C. The sections were then incubated with Alexa Fluor® 488 Conjugate (ZSGB-BIO, #ZF-0512, 1:100, China) and Alexa Fluor® 594 Conjugate (ZSGB-BIO, #ZF-0513, 1:100, China) secondary antibodies at room temperature for 2 h. The results were investigated under an immunofluorescence microscope.
4
Mitochondrial Protein Localization Visualization
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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.
5
Protein Expression Analysis Protocol
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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.
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