Proteins in cell lysates were detected with antibodies against phospho-PAK1 (Santa Cruz Biotechnology), PAK1, phospho-AKT, AKT, HIF1α (BD Biosciences, North Ryde, Australia), and GAPDH. Antibodies were from Cell Signalling Technology (Arundel, Australia), unless otherwise stated. Bound antibodies were visualized using ECL reagents (GE Healthcare, Amersham, UK), and the density of each band was analysed using Multigauge computer software (Berthold, Bundoora, Australia). HIF1α expression was determined in cells cultured under normoxia or hypoxia (1 % O2).
Multigauge computer software
Manufactured by Berthold Technologies
Sourced in Australia
Multigauge is a computer software application developed by Berthold Technologies for data acquisition and analysis. It is designed to work with a variety of laboratory equipment and sensors to collect, process, and display measurement data.
Automatically generated - may contain errors
Lab products found in correlation
Ecl reagent, by GE Healthcare (4 mentions)
Hif 1α, by BD (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Phospho pak1, by Santa Cruz Biotechnology (1 mentions)
Phospho akt, by BD (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Sigmaplot 12, by IBM (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Ab9643, by Abcam (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab17157, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
6 protocols using multigauge computer software
1
Analyzing Protein Signaling in Cells
2
β-Catenin Immunoprecipitation and Immunoblotting
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Cells were lysed in lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl2, 1% NP40, 1 mmol/L DTT, 5 μg/mL aprotinin, 5 μg/mL leupeptin, and 1 mmol/L PMSF). The cell lysates were centrifuged at 13,000 g for 10 min at 4°C, and the protein concentration from the resultant supernatants was quantified using Bradford reagent (Sigma). In some cases, the supernatants were immunoprecipitated with anti‐β‐catenin antibody, followed by immunoblotting. For immunoblotting samples from either cell lysates or immunoprecipitates were resolved by SDS‐PAGE, and proteins were detected with the antibodies indicated in the text. The bound antibodies were visualized using ECL reagents (GE Healthcare, Amersham, Buckinghamshire, UK), and the density of each band was analyzed using Multigauge computer software (Berthold, Bundoora, Vic., Australia).
3
Protein Expression Analysis in CRC
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Total protein was extracted form CRC tumor tissues, corresponding adjacent normal tissues and cultured cells. The protein concentration was determined by the BCA method. 40µg total proteins were separated by SDS-PAGE, followed by transferring to a PVDF membrane (GE Healthcare). Then the membranes were blocked with 5% skimmed milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T), for 1 h and subsequently incubated overnight at 4 °C with the primary antibodies, including SLPI (#sc373802, 1:200, Santa Cruz biotechnology, Santa Cruz, CA), Phospho-AKT (#9271, 1:1000, Cell Signaling Technology) and AKT (#9272, 1:1000, Cell Signaling Technology), and GAPDH (1:1000, #sc32233, Santa Cruz Biotechnology, Santa Cruz, CA). After that, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies IgG (Sigma, 1: 5,000) for 2 h at room temperature. The protein bands were visualized with Amersham ECL substrates and analyzed by Multigauge computer software (Berthold, Bundoora, Australia).
4
Protein Expression Analysis in APC Mice
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Proteins were extracted from tumours and from adjacent normal tissues of small intestine and colon/rectum of APC∆14/+ mice using the method described previously [5 (link), 14 (link)]. Proteins were separated by running the samples through 10% SDS-PAGE, and then blotted with antibodies against Bmi1 (Gene Tex, Irvine, CA), PAK1 or GAPDH (Cell Signaling, Danvers, MA). Bound antibodies were visualized using ECL reagents (GE Healthcare, Amersham, UK), and the density of each band was analysed using Multi-gauge computer software (Berthold). The correlation of Bmi1 and PAK1 was analysed using the program Sigma Plot 12 (SPSS, Chicago, IL).
5
Protein Expression Analysis in Xenograft Tumors
6
Protein Quantification and Western Blot Analysis
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The complete protein was extracted and quantified using the BCA method. We used the SDS-PAGE to collect 40 µg of total protein and transferred it to a PVDF membrane (GE Healthcare). Therefore, the membrane sealing, and antibody incubation were conducted with 5% skimmed milk powder at room temperature for 1 h. Subsequently, the antibodies were diluted into the blocking solution following the manuscript instructions and incubated with the membrane at 5 ℃ overnight. The primary antibodies included β—actin first antibody (beyotime), SLPI first antibody (Abcam, ab17157), PUMA first antibody (Abcam, ab9643) and p-foxo3 α first antibody (Abcam, ab9643), Ab47285) FoxO3 α first antibody (Abcam, ab12162) p65 (Abcam, ab16502) p-p65 (Abcam, ab76302) p-Akt (Abcam, ab81283) Akt (Abcam, ab8805) Bax (Abcam, ab32503) c-caspase3 (Abcam, ab2302) Ki67 (Abcam, ab15580). Furthermore, we used the TBST to wash the membrane incubated with a primary antibody three times, 10 min each time. Then, secondary antibody second antibody (beyotime) was diluted and incubated with the membrane at room temperature for 2 h, washed with TBST three times, 10 min each time before the bands' observation through Amersham ECL solution, and assessed with Multi gauge computer software (Berthold, Bundoora, Australia).
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