Total BM EVs or nucleated cells were lysed in NuPAGE LDS lysis buffer (Life Technologies) and proteins were quantified using the DC protein assay (Biorad). 20ug total proteins were loaded per lane. Immunoblotting was performed using rabbit polyclonal anti-GFP (ab290-abcam) and rabbit monoclonal anti-TSG101 (EPR7130B-abcam).
Nupage lds lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NuPAGE-LDS lysis buffer is a sample preparation solution designed for protein extraction and solubilization. It is used to prepare protein samples for analysis using NuPAGE gel electrophoresis systems.
Automatically generated - may contain errors
Lab products found in correlation
Ab290, by Abcam (1 mentions)
Epr7130 b, by Abcam (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Sc 11769, by Abcam (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Cpt1a, by Proteintech (1 mentions)
Mcl 1, by Affinity Biosciences (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
P akt, by Abcam (1 mentions)
P egfr, by Abcam (1 mentions)
Raptor, by Proteintech (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
5 protocols using nupage lds lysis buffer
1
Protein Quantification and Immunoblotting
2
TGF-β Signaling Pathway Analysis
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For pSmad3 blots, to confirm TGF-β inhibitors performed as expected, cells were serum starved for 1 hour in GMEM then placed in fibroblast media containing 10 ng/ml TGF-β (R&D Systems) with or without TGF-β inhibitors, then western blotting was performed as for all experiments, as follows. Cells were harvested in Trypsin-EDTA, lysed with 1x Nupage LDS lysis buffer (Life Technologies), with or without phosphatase inhibitors (HALT, Life Technologies), heated to 95°C for 10 minutes followed by sonication of 3 cycles of 15 s on the Misonix XL2000 sonicator on setting 2. Protein concentration was measured by BCA assay (Life Technologies), and 1-100 μg lysate was mixed with DTT (final 100 μM), then run at 150 V and transferred at 50 V for 3-hours with BioRad Mini-PROTEAN and Mini Trans-Blot Cell tanks respectively. Antibodies used included pSMAD2/3 antibody (Santa Cruz sc-11769), and B-ACTIN-HRP antibody (Abcam, ab20272l).
3
Cellular Biomarker Profiling Assay
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For standard assay of inhibitor effects on cellular biomarker expression, cells were plated at pre-determined density in 6-well or 12-well culture plates for overnight, treated with inhibitors for the indicated doses and times. Cells were lysed in NuPAGE-LDS lysis buffer (Invitrogen) and immunoblotted with antibodies including P-S6K1(T389), S6, P-4EBP1(T70), 4EBP1, P-GSK3(S21/9), GSK3, cyclin D1, Raptor, Rictor, P-ACL(S455), ACL, cleaved-PARP (Cell Signaling Technology); P-S6(S235/6), AKT(S473), AKT, Bim (Abcam-Epitomics); c-Myc (Santa Cruz); Mcl-1 (Affinity); CPT1A (Proteintech); β-actin and GAPDH (Bioworld).
4
Investigating EGF-Mediated Signaling Pathways
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Cells were seeded at appropriate density 1 day before drug treatment. Cells stimulated by EGF were starved with serum-free culture medium overnight. Various inhibitors (taken from 20 mM stock in DMSO) were first prepared as 100x concentrated solutions before being added to cell culture. Cells were treated for 24 h or as indicated, lysed in NuPAGE-LDS lysis buffer (Invitrogen) and immunoblotted with primary antibodies against TF (R&D, Cat#MAB2339), P-EGFR (Epitomics, Cat#1138-1), Raptor (Proteintech, Cat#20984-1-AP), Rictor (CST, Cat#2114), P-S6 (Epitomics, Cat#2268-1), P-AKT (Abcam, Cat#Ab81283), pERK (CST, Cat#4370), and GAPDH (Bioworld, Cat#AP0063).
5
Western Blot Protein Quantification
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For the standard assay of inhibitory effects, cells plated at predetermined density in 12-well culture plates were treated with cyy-287 or gefitinib at indicated doses for indicate time, lysed in NuPAGE-LDS lysis buffer (Invitrogen, Carlsbad, USA). The protein samples were then subject to 10% SDS-PAGE and then transferred to a 0.45 μM nitrocellulose filter membranes (PALL, New York, USA). Membranes were blocked at room temperature with freshly prepared 5% skim milk powder for 2.5 h, followed by incubation with various antibodies (1:1000) as indicated overnight at 4°C. Then membranes were incubated with the corresponding HRP-conjugated secondary antibody (1:10,000; Jackson, West Grove, USA) for 2 h. After washing by TBST for three times, protein bands on membranes were detected using an enhanced chemiluminescence detection kit (Millipore, Billerica, USA) and the protein gray value was quantified by ImageJ software (
https://imagej.nih.gov/ij/ ). GAPDH was used as the loading control.
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