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5 protocols using hrp conjugated anti mouse and anti rabbit igg

1

Western Blot Analysis of Cell Signaling

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The WB followed the previous research procedure [22 (link)]. The HK-2 cells pretreated with different bacteria were lysed in RIPA buffer. The cell proteins were extracted for quantitative denaturation, and then detected by WB. SDS-polyacrylamide gel used to separate proteins and then transferred to NC membrane (Millipore). Antibodies of CD44, p-P38, P38, p-P65, and P65 were immunoblotted with an NC membrane. HRP-conjugated anti-mouse and anti-rabbit IgG (sc-2005 and sc-2004, Santa Cruz) are secondary antibodies. Internal control was tubulin. ImageJ software was used to quantify and normalize processing.
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2

Investigating Cellular Signaling Pathways

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HGF was procured from Peprotech and c-MET inhibitor (SU11274) from Santa Cruz. Primary antibodies were purchased from Abcam (hTERT), Cell Signalling Technology (Phospho-c-MET, vimentin, E-Cadherin, N-Cadherin), Santa Cruz (c-MET), Chemicon (Cytokeratin-18a), and Thermo Fisher Scientific (β-Actin). Secondary antibodies, HRP conjugated anti-mouse and anti-rabbit IgG, and HRP conjugated anti-mouse IgM were obtained from Santa Cruz. Secondary antibodies, FITC conjugated anti-mouse and anti-rabbit IgG were obtained from Santa Cruz.
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3

Western Blot Analysis of Autophagy and Oxidative Stress Markers

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Western blot analysis was conducted as described previously. Briefly, the cells were lysed in RIPA buffer, and equal amounts of protein were separated on an SDS polyacrylamide gel, transferred to an NC membrane (Millipore) and then immunoblotted with antibodies. Primary antibodies against the following proteins were used in this study: Beclin1(#3495), SQSTM1/P62 (#5114), LC3A/B (#12741), phospho-p38 (T180/Y182, #4511) and p38 (#9212) were purchased from Cell Signaling Technology, 8-OHdG (ab10802) and SOD1 (ab51254) were purchased from Abcam, GAPDH (sc-365062) was purchased from Santa Cruz. The following secondary antibodies used in the study were purchased from Santa Cruz: HRP-conjugated anti-mouse and anti-rabbit IgG (sc-2005 and sc-2004). The band intensities were quantified and normalized to the band intensities of GAPDH using ImageJ software. The data were presented as bar graphs after their statistical validity was tested.
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Evaluation of Epithelial-Mesenchymal Transition Markers

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After incubation in normoxia or for 6, 12, or 24 h under hypoxia, cells were lysed and the protein concentration was determined (10). Proteins were separated on 10% polyacrylamide gels and transferred onto a nitrocellulose membrane. The monoclonal antibodies used for blotting were: anti-N-cadherin (mouse, 1:3000), anti-E-cadherin (mouse, 1:10,000) and anti-GAPDH (mouse, 1:100,000) (all from BD Transduction, San-Jose, CA, USA), anti-vimentin (mouse, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Snail (rabbit, 1:500; Novus Biologicals, Little Town, CO, USA), anti-Twist (mouse, 1:50; Abcam, Cambridge, UK) and HRP-conjugated anti-rabbit and anti-mouse IgG (both 1:7000; Santa Cruz Biotechnology). Western blot signals were measured by densitometric scanning with an Azure C300 Intelligent Dark Box (Biosystems Inc., Dublin, CA, USA). Each Western blot was performed three times.
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5

Protein Expression Analysis in Tissue Biopsies

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Patient-derived tissue biopsies and harvested cells were lysed in RIPA buffer (Beyotime) for 30 min on ice. The lysate was centrifuged at 20,000 ×g for 20 min at 4 °C. The supernatant was transferred, and protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of total and nuclear proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and electro-transferred onto a nitrocellulose membrane. Membranes were incubated with antibodies to NHE1 (Santa Cruz Biotechnology), CyclinD1 (Abcam, Cambridge, UK), PCNA (Abcam), MMP-2 (Santa-Cruz), MMP-9 (Santa Cruz Biotechnology), ERK1/2 (Epitomics, Burlingame, CA), phospho-ERK1/2 (Epitomics). The secondary antibodies used for detection were HRP-conjugated anti-rabbit and anti-mouse IgG (Santa Cruz Biotechnology). Immunoreactive bands were detected by an enhanced chemiluminescence kit (EMD Millipore, Darmstadt, Germany) and results were quantitated using an image analyzer Quantity One System (Bio-Rad Laboratories, Hercules, CA). β-actin and LaminB (Santa Cruz Biotechnology) were used as loading control for total and nuclear fractions, respectively.
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