Western blot analysis was completed as previously described (MacDougall et al., 2017 (link)). Briefly, total cell proteins were separated by SDS-PAGE, transferred to PVDF membranes and analysed using the Gel Doc EZ imager (Bio-Rad). Membranes were blocked in TBS-Tween containing ovalbumin (0.1%), followed by incubation with primary antibodies (pERK, Total-ERK, and CD147 Santa Cruz, USA; pAKT, total AKT and IκB Cell Signalling Technology, USA; β-tubulin, Abcam, United Kingdom; CypA, Biomol, Germany; all antibodies at 1:5000 dilution) overnight at 4 °C with gentle rocking. Membranes were washed and probed with anti-mouse or anti-rabbit IgG horseradish peroxidase conjugated secondary antibodies (GE Lifesciences, USA). Immunoreactive bands were visualized using ECL Plus (Amersham) chemiluminescent detection reagent and quantified by densitometry. Total loaded protein levels in gel lanes were also determined by visualising the loaded proteins on TGX stain free gels (Bio-Rad) using the Gel Doc EZ imager (Bio-Rad).
Cd147
Manufactured by Santa Cruz Biotechnology
Sourced in United States
CD147, also known as Basigin or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. It functions as a cell surface receptor involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
β3gnt8, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Total erk, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Abcam (1 mentions)
Tgx stain free gel, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg secondary antibodies, by GE Healthcare (1 mentions)
Ecl plu, by Cytiva (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
Catalase, by Abcam (1 mentions)
α tubulin, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Tritc, by Jackson ImmunoResearch (1 mentions)
Acox1, by Santa Cruz Biotechnology (1 mentions)
Acox3, by Santa Cruz Biotechnology (1 mentions)
Pex14, by Merck Group (1 mentions)
Pmp70, by Merck Group (1 mentions)
Galectin 3, by Abcam (1 mentions)
Secondary anti mouse igg antibody, by Abcam (1 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
5 protocols using cd147
1
Western Blot Analysis of Protein Signaling
2
Immunofluorescence and Western Blotting for Metabolic Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the immunofluorescence experiments, the following antibodies were used: MCT2 (sc-14926; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT1 (sc-365501; Santa Cruz Biotechnology), MCT4 (sc-50329; Santa Cruz Biotechnology), CD147 (sc-71038; Santa Cruz Biotechnology), gp70 (HPA017740, 1:100; Atlas Antibodies, Stockholm, Sweden), PEX14 (a gift from Dr. Dennis Crane, Griffith University, Brisbane, Australia), catalase (ab88650; Abcam, Cambridge, UK), TRITC (Jackson ImmunoResearch, West Grove, PA, USA) and Alexa 488 (Invitrogen, Life Technologies, Carlsbad, CA, USA). For the Western blot analysis, the following antibodies were used: MCT2, ACOX1 (a gift from A. Völkl, University of Heidelberg, Germany), ACOX3 (sc-135435; Santa Cruz Biotechnology), PEX14, PMP70 (SAB4200181; Sigma-Aldrich, St Louis, MO, USA) and α-Tubulin (T9026, Sigma-Aldrich). For the immunohistochemistry (IHC) staining, the following antibodies were used: AMACR (504R-16, Cell Marque, Rocklin, CA, USA), ACOX3 (sc-135435; Santa Cruz Biotechnology) and DBP (a gift from Dr. Gabriele Moller from HelmholtzZentrum München). Pex19-YFP plasmid was a gift from Dr M. Schrader, Exeter University, UK.
3
Colorectal Cancer Tissue Microarray Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue microarray slides were obtained from Outdo Biotech (Shanghai, China), which contained 90 pairs of adjacent paracancer tissues and colorectal cancer tissues. The slides were stained with primary antibodies against β3GnT8, CD147 (Santa Cruz Biotechnology, Dallas, TX, United States), galectin3 (Abcam, Cambridge, MA, United States), MMP-2 (Abcam), β3GnT2 (Santa Cruz Biotechnology), and HRP-conjugated anti-rabbit IgG, or anti-mouse IgG secondary antibody (Abcam). The protein expression was detected by DAB horseradish peroxidase color development kit (Beyotime, Haimen, China). The slides were evaluated by the staining intensity and positive cells percentage as follows: staining intensity, 0(no), 1(weak), 2(moderate), 3(strong), and positive cells percentage, 0(<1%), 1(1–33%), 2(34–66%), 3(67–100%). The final grade of target protein expression was calculated by plus the score of staining intensity and the score of positive cells percentage: 0(0), 1+(1–2), 2+(3–4), and 3+(5–6). The expression scores of β3GnT8, CD147, galectin3 and MMP-2 were provided in Supplementary Data Sheet 1 (Supplementary Tables 1–4 ).
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were harvested and were resolved by SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk or 1% BSA in TBST at room temperature for 2 h, followed by an overnight incubation with primary antibody against β3GnT8, CD147 (Santa Cruz, Dallas, TX, USA), TIMP2 (Santa Cruz, Dallas, TX, USA), c-jun (Abcam, Cambridge, MA, USA) or GAPDH (Beyotime, Haimen, China) at 4°C. The rabbit anti-human β3GnT8 affinity polyclonal antibody was produced by our laboratory as previously described [20 (link)]. Following 3 washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime, Haimen, China) at room temperature for 1 h. After additional 3 washes with TBST, the protein bands were visualized using an enhanced chemiluminescence (ECL) kit (GE Healthcare Life Sciences, Shanghai, China).
5
Gene Expression and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from each experimental group of cells was extracted using TRIzol (Gibco-BRL) according to the manufacturer's instructions. cDNA was generated from total RNA using M-MLV Reverse Transcriptase (Fermentas, USA). Amplification was performed for >28 cycles. PCR products were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide to visualize the bands. Primer sequences and expected product sizes are listed in Table I.
Western blot analysis. Western blot analysis was performed as previously described (23, 24) . In brief, the cells were lysed with lysis buffer and 40 µg of protein from each sample was resolved by 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was blotted with antibodies β3Gn-T8, GAPDH, c-jun and CD147 (all purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Western blot analysis. Western blot analysis was performed as previously described (23, 24) . In brief, the cells were lysed with lysis buffer and 40 µg of protein from each sample was resolved by 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was blotted with antibodies β3Gn-T8, GAPDH, c-jun and CD147 (all purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!