Cell lysates subjected to SDS polyacrylamide electrophoresis on pre-cast 4–20% gradient gels (GenScript) and separated proteins transferred to PVDF immunoblotting membrane (Bio-Rad). PVDF membrane was blocked in 5% w/v skim milk (from powder, EMD Millipore), and stained with primary antibodies and secondary horseradish peroxidase-conjugated antibody (see Antibodies, above). Signal was developed with ECL western blotting substrate (Pierce Chemical) and captured on x-ray film. For re-probing with additional antibodies, blots were stripped (Re-Blot Plus, Millipore) and blocked again with skim milk, as above. Blots were scanned digitally and bands were quantified with Image J. All experiments were repeated three times with similar results.
Gradient gel
Manufactured by GenScript
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4–20% gradient gels are laboratory equipment used for protein separation and analysis. These gels feature a continuous gradient of polyacrylamide concentrations, ranging from 4% to 20%, which allows for the effective separation of a wide range of protein sizes in a single run.
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Lab products found in correlation
Re blot plus, by Merck Group (2 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Pvdf immunoblotting membrane, by Bio-Rad (2 mentions)
Quickblock western blocking buffer, by Beyotime (2 mentions)
Anti ha, by Roche (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti occludin antibody, by Abcam (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Anti zo 1 antibody, by Proteintech (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ap307p, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent, by Thermo Fisher Scientific (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
14 protocols using gradient gel
1
Western Blot Protein Analysis
2
Isolation and Separation of Ciliary Ectosomes
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Ciliary ectosomes harvested from 1.2 × 108 adhering gametes were mixed with freshly washed minus gametes (4 × 107 cells) in 0.4 ml N-free medium with gentle agitation. After 15 min, after removing a portion to be used as input, a portion was centrifuged at 20,000×g for 2 min to sediment whole cells and any bound vesicles. The remaining portion was de-ciliated, and centrifuged at 600×g for 2 min to sediment the cell bodies. The supernatant was centrifuged at 20,000×g for 2 min to sediment the cilia. All the fractions were boiled in 1 × SDS sample buffer for 5 min, and then subjected to SDS/PAGE analysis on 4–20% gradient gels (GenScript, USA).
3
Quantitative Immunoblotting of Ciliary Proteins
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SDS-PAGE and immunoblotting were essentially as described previously (Cao et al., 2013 (link)) with slight modifications (Belzile et al., 2013 (link)). Cells, cilia, and ciliary ectosome samples were boiled in 1 × SDS sample buffer for 5 min, and then subjected to SDS-PAGE analysis on 4–20% gradient gels (GenScript, USA). The antibodies used for immunoblotting were anti-HA (1:1000; Roche), anti-α-tubulin (1:3000 or 1:200,000; Sigma), anti-GSP1 (1:20,000; Wilson et al., 1999 (link)), anti-FMG1 (1:100,00), anti-IFT139 (1:50,000), anti-IFT81 (1:1000), and anti-BUG22 (1:500,000). Mouse monoclonal antibody against FMG1 was generously provided by Robert Bloodgood (University of Virginia). Monoclonal antibodies against IFT81 and IFT139 were generously provided by Dennis Deiner and Joel Rosenbaum (Yale University). Rabbit polyclonal antibody against BUG22 was generously provided by Dan Meng and Junmin Pan (Tsinghua University). The amount of SAG1-HA released into the medium during adhesion was determined by use of ImageJ analysis of immunoblots of equal proportions of cells and medium. Determinations were made under conditions in which the amount of sample loaded was linear with the signal obtained. Similar methods were used to determine the relative abundance of FMG1 and SAG1-HA in equal protein amounts of ciliary ectosomes compared to cilia.
4
Protein Separation and Western Blotting
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Cell lysates subjected to SDS polyacrylamide electrophoresis on pre-cast 4–20% gradient gels (GenScript) and separated proteins transferred to PVDF immunoblotting membrane (Bio-Rad). PVDF membrane was blocked in 5% w/v skim milk (from powder, EMD Millipore), and stained with primary antibodies and secondary horseradish peroxidase-conjugated antibody (see Antibodies, above). Signal was developed with ECL western blotting substrate (Pierce Chemical) and captured on x-ray film. For re-probing with additional antibodies, blots were stripped (Re-Blot Plus, Millipore) and blocked again with skim milk, as above.
5
Quantitative Protein Analysis Pipeline
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Extracted proteins were quantified using a BCA Protein Assay kit (Cat# 23,227, Thermo Fisher Scientific), and the samples were loaded onto SDS-PAGE using 4–20% gradient gels (Cat# M00656, GenScript Biotech), followed by transfer to a PVDF membrane (Cat# ISEQ00010, Merck Millipore). After the transfer, the membrane was blocked with QuickBlock™ Western Blocking Buffer (Cat# P0252, Beyotime Biotech) for 1 h at room temperature and then used for immunoblotting. Antibodies used in study included an anti-occludin antibody (Cat# ab167161, Abcam), an anti-ZO-1 antibody (Cat# 21773-1-AP, Proteintech), and an anti-β-actin antibody (Cat# 66009-1-1 g, Proteintech), an anti-TSP1 antibody (Cat# sc-59,887, Santa Cruz Biotechnology), and an anti-transferrin antibody (Cat# ab278498, Abcam).
6
Western Blot Analysis of Proteins
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Total proteins were prepared utilizing M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) and histone was isolated by Histone Extraction Kit (Bestbio, China). Proteins were separated by SDS-PAGE on 4–20% gradient gels (GenScript, USA) and then transferred onto 0.22 μm PVDF membrane (Millipore, USA). After blocking with QuickBlock™ Western Blocking Buffer (Beyotime, China), the membranes were probed with the primary antibodies for 4°C overnight and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:10,000; AP307P, Millipore, USA) for 1 h at room temperature. The luminescence signal was visualized using the SuperSignal West pico chemiluminescent (Thermo Fisher Scientific, USA) and captured by digital chemiluminescence imager (Bio-Rad, USA). GAPDH or Histone H3 was applied as endogenous reference for total protein and histone. The primary antibodies used in this study were listed in Supplementary Table S2
7
Analyzing EGFR Signaling Dynamics
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Cells were seeded at a nonconfluent density on 10-mm plates coated with collagen I. Cells were washed with ice-cold PBS, and cellular protein in treated and untreated samples was extracted using a 150 μl of RIPA buffer on the dish 10, 30, and 60 min after DMSO removal. Cellular extracts were kept on ice for 5 min, and 5× Laemmli buffer was added. The samples that were blotted for ppMRLC2 contained an additional 1 mM ATP. Extracts were boiled for 15 min before loading on 4–12% gradient gels (GenScript, Piscataway, NJ). Wet transfer was performed onto polyvinylidene fluoride membranes. The primary antibodies ppMRLC2, pEGFR (Tyr-992), EGFR, α-tubulin, and β-actin (all Cell Signaling) were used at 1/1000 concentration in 5% milk in PBS/Tween-20 (0.02%). The secondary antibody anti-rabbit (Cell Signaling) was used at 1/2000 (Cell Signaling) and anti-mouse (Bethyl, Montomery, TX) at 1/5000 in PBS.
8
Affinity Purification of Flag-Slug Protein Complex
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HEK293T cells expressing Flag-Slug were lysed in lysis buffer (50 mM Tris–HCl, pH 8.0, 0.2% NP-40, 150 mM NaCl, 2 mM EDTA and 1 × complete protease inhibitor cocktail) on ice for 30 min and centrifuged at 10,000 g for 10 min at 4 °C to collect the supernatant. Cell lysates extracted from about 5 × 108 cells were incubated with 150 µL equilibrated anti-Flag M2-agarose beads for 1.5 h at 4 °C. After binding, the beads were washed with cold lysis buffer five times. Flag peptide (Sigma) was added into the resin to elute the Flag protein complex overnight at 4 °C and centrifuged at 8,000 g to collect the supernatant. Then the supernatant was resolved on 4–12% gradient gels (GenScript), stained using silver stain kit (Pierce), and subjected to LC–MS/MS (ThermoFisher Q Exactive mass spectrometer) sequencing.
9
Western Blot Analysis of Stress Proteins
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Western Blotting was performed according to the Cold Spring Harbor protocol. Briefly, cells were lysed in a RIPA buffer (Boston BioProducts, MA, USA) using 25-gauge syringes. The same protein amounts were subjected to 4–20% gradient gel (Genscript, NJ, USA), followed by transfer to a polyvinylidene fluoride (PVDF) membrane using wet methods where appropriate. The membranes were blocked in blocking solution (LI-COR, Inc., Lincoln, NE, USA) for 60 min unless otherwise specified, and incubated overnight with a mouse monoclonal anti-HSP70 antibody (1/1000, SMC100, StressMarq Biosciences, BC, Canada), a rabbit polyclonal anti-BAG3 antibody (1/1000, 10599-1-AP, Proteintech, IL, USA), a mouse monoclonal anti-HSP90α antibody (1/1000, SMC147, StressMarq, Biosciences, BC, Canada) and a rabbit polyclonal anti-GAPDH antibody (1/1000, sc-25778, Santa Cruz, CA, USA). The membranes were incubated for 1 h at room temperature with goat anti-rabbit IRDye 800 CW fluorescent secondary antibodies (1/15,000, LI-COR, Inc., Lincoln, NE, USA) and 680 RD fluorescent secondary antibodies (1/15,000, LI-COR, Inc., Lincoln, NE, USA). Blots were washed with Tris buffered saline, 0.1% (w/v) Tween 20 (TBS-T) and visualized with the Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA). The quantitative densitometric analysis was performed using Image Studio Lite Ver. 5.2 (LI-COR, Inc., Lincoln, NE, USA).
10
Whole Protein Quantification on PVDF Membrane
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Protein samples (5 μg each) were loaded on 4–20% gradient gel (Genscript, NJ, USA). After the electrophoresis run, the gel transferred to PVDF membrane using wet methods. The membrane was hydrated using 100% methanol for 30 s and washed with tris-buffered saline, pH7.4 (TBS) for 5 min at room temperature with gentle shaking. The membrane was stained with Revert 700 total protein stain solution (LI-COR, Inc., Lincoln, NE, USA) and incubated for 5 min at room temperature with washing solution (LI-COR, Inc., Lincoln, NE, USA). The membrane was then washed with Revert 700 Wash Solution n (LI-COR, Inc., Lincoln, NE, USA), rinsed two times for 30 s at room temperature with gentle shaking. The membrane was then briefly rinse with ultrapure water. For visualizing the membrane, the membrane was imaged in the 700 nm channel using Image Studio Lite Ver. 5.2 (LI-COR, Inc., Lincoln, NE, USA).
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