Protein samples were transferred to polyvinylidene difluoride membrane (ISEQ00010, Millipore). The blots were blocked with 5% nonfat milk for 2 h and then incubated with primary antibodies and secondary antibodies. The Clarity Western ECL Substrate Kit (170-5061, Bio-rad) was used to visualize the immunoreactive bands. Images were captured with an Image Quant LAS4000 system (GE Healthcare Life Sciences, Piscataway, NJ, USA). β-actin served as a protein loading control. The following antibodies were used: BAX (A0207, ABclonal; 1:1000), BCL2 (60178-1-Ig, 1:3000; Proteintech,), CDC42 (ab187643, Abcam; 1:20000), Cofilin (A1704, ABclonal; 1:1000), IQGAP1 (sc-376021, Santa Cruz; 1:500), LIMK1 (ab108507, Abcam; 1:5000), N-cadherin (33-3900, Invitrogen; 1:500), Occludin (71–1500, Invitrogen; 1:500), PAK1 (A19608, ABclonal; 1:1000), PAFAH1B1 (ab109630, Abcam; 1:5000), PCNA (A12427, ABclonal; 1:1000), p-Cofilin (AP0178, ABclonal; 1:1000), ZO-1 (61-7300, Invitrogen; 1:500), β-actin (AC028, ABclonal; 1:100000), β-catenin (71-2700, Invitrogen; 1:500), HRP Goat Anti-Mouse IgG (AS003, ABclonal; 1:3000), and HRP Goat Anti-Rabbit IgG (AS014, ABclonal; 1:3000).
Sc 376021
Manufactured by Santa Cruz Biotechnology
Sourced in China
Sc-376021 is a laboratory product manufactured by Santa Cruz Biotechnology. It is a research-use-only tool designed for specific scientific applications. The core function of this product is to facilitate specific research activities. No further details about its intended use or application can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab32575, by Abcam (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Ab187643, by Abcam (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Clarity western ecl substrate kit, by Bio-Rad (1 mentions)
Odyssey v3, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Hc101 01, by Transgene (1 mentions)
Goat anti mouse irdye 800 conjugated secondary antibody, by LI COR (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab8245, by Abcam (1 mentions)
4 protocols using sc 376021
1
Western Blot Analysis of Protein Expression
2
Immunohistochemical Analysis of Liver Cells
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Liver samples were fixed in 4% paraformaldehyde for 48 h. After being dehydrated and embedded, the samples were cut into 5-μm slices. For preparation of hepatocytes and NPCs smear, isolated cells were fixed in 4% paraformaldehyde for 1 h. After permeabilization in PBS containing 0.5% Triton X-100 for 15 min, BMSCs were blocked with 2% BSA for 30 min. Primary antibodies of IQGAP1 (1:100; sc-376021, Santa Cruz Biotechnology, CA), α-SMA (1:100; ab32575, Abcam, Bristol, UK), and albumin (1:100, Santa Cruz Biotechnology) were used. Fluorescein isothiocyanate (FITC)- or Cy3-conjugated AffiniPure Goat Anti-Rabbit or -Mouse IgG (1:100; Jackson ImmunoResearch, PA) was used as secondary antibody. Nuclei were stained with DAPI. The samples were observed under a confocal microscope (LSM510, Carl Zeiss MicroImaging, Jena, Germany).
3
Immunohistochemistry Analysis of IQGAP1
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Immunohistochemistry was performed as previously described[19 (link)] with mouse antibody to IQGAP1 (1:100, Santa Cruz sc-376021). Secondary staining with biotinylated horse secondary to mouse IgG at 1:1000 for 1hour (Vector Laboratories). Negative control samples were stained using the same protocol without addition of primary antibody. Antibody was tested for specificity using cell lines with IQGAP1 depletion using CRISPR-Cas9 as described above.
4
Western Blot Analysis of BMSC Markers
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BMSCs were cultured in 10-cm tissue culture plates and treated with corresponding reagents. Radio-immunoprecipitation assay (RIPA) lysis buffer (R0010, Solarbio, Beijing, China) was used to prepare lysates. Fifty to one hundred micrograms of protein extracts were electrophoresed in 8% (for IQGAP1) or 12% (for α-SMA) SDS/polyacrylamide gels and transferred the protein to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat milk at room temperature for 1 h. The blots were incubated with primary antibodies against IQGAP1 (1:1,000; sc-376021, Santa Cruz Biotechnology) or α-SMA (1:1,000; ab32575, Abcam), β-tubulin (1:5,000; HC101-01, Transgen Biotech, China), GAPDH (1:1,000; ab8245, Abcam), Cdc42 (1:1,000; 16,119, Thermo Scientific), Rac1 (1:167; 16,118, Thermo Scientific), or actin (1:2,000; Abcam). After 1 h incubation at room temperature with goat anti-rabbit IRDye-800-conjugated secondary antibody, goat anti-rabbit IRDye-700-conjugated secondary antibody, goat anti-mouse IRDye-800-conjugated secondary antibody, and goat anti-mouse IRDye-700-conjugated secondary antibody (1:10,000; 926–32,211, 926–68,021, 926–32,210, and 926–68,020, LI-COR Biosciences, Lincoln, NE), the signals were displayed using Odyssey Imaging System and quantified by the Odyssey v3.0 software (LI-COR Biosciences).
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