The following were purchased from Sigma-Aldrich (Dorset, UK): Dulbecco’s Modified Eagle Medium (DMEM), Penicillin/Streptomycin, MEM non-essential amino acids 100×, 0.25% Trypsin-EDTA, L-glutamine, putrescine, spermidine, spermine. Fetal bovine serum (FBS) was from Gibco by Life Technologies (Sao Paolo, Brazil), mTOR siRNA and negative control were from Fisher Chemical (Loughborough, UK), protease and phosphatase inhibitors cocktail from Roche (Indianapolis, IN, USA), BCA protein assay kit from BioVision (Milpitas, CA, USA). RIPA lysis buffer 10× from Millipore (Burlinton, MA, USA). The following antibodies were purchased from Abcam Biotechnology (Cambridge, UK) and used at the indicated dilutions: rabbit polyclonal anti-actin antibody (ab119716, 1:7, 500 dilution), goat polyclonal anti-rabbit IgG-HRP (ab205718, 1:10,000 dilution). Additionally, mouse monoclonal anti-ODC antibody (sc-398116, 1:100 dilution), mouse monoclonal anti-phospho-4EBP1 (sc-293124, 1:200 dilution), mouse monoclonal anti-phospo-p70S6K (sc-8416, 1:300 dilution), mouse monoclonal anti- eIF4E (sc-271480, 1:300 dilution) and goat anti-mouse IgG-HRP (sc-2005, 1:2,500 dilution) antibodies were from Santa-Cruz Biotechnology (Heidelberg, Germany). Rapamycin and the class 1 dual inhibitors of PI3K and mTORC1, NVP-BEZ235, were from Cayman Chemicals (Ann Arbor, MI, USA).
Ripa lysis buffer 10
Manufactured by Merck Group
Sourced in United States
RIPA lysis buffer 10x is a concentrated solution used to extract and solubilize cellular proteins for analysis. It contains a balanced mixture of ionic and non-ionic detergents, as well as other components to facilitate efficient cell lysis and protein extraction.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Goat polyclonal anti rabbit igg hrp, by Abcam (2 mentions)
Protease and phosphatase inhibitors cocktail, by Roche (2 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (2 mentions)
Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Nvp bez235, by Cayman Chemical (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Rabbit anti actin polyclonal antibody, by Abcam (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Spermidine, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
Donkey anti goat igg h l hrp, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey scanner and software, by LI COR (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
7 protocols using ripa lysis buffer 10
1
Regulation of mTOR Signaling Pathway
2
Polyamine Signaling Pathway Analysis
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The following were purchased from Sigma-Aldrich (Dorset, UK): Dulbecco’s Modified Eagle Medium (DMEM), Penicillin/Streptomycin, MEM non-essential amino acids 100×, Trypsin-EDTA, L-glutamine, putrescine, spermidine, and spermine. Foetal bovine serum (FBS) was from Gibco Life Technologies, Sao Paolo, Brazil. The protease and phosphatase inhibitors cocktail were from Roche, Indianapolis, IN, USA. RIPA lysis buffer 10× from Millipore, Burlington, VT, USA. The following antibodies were purchased from Abcam Biotechnology, Cambridge, UK: rabbit polyclonal anti-β-actin (ab119716, 1:7500 dilution), goat polyclonal anti-rabbit IgG-HRP (ab205718, 1:10,000 dilution), goat polyclonal anti-AZ1 (ab223481, 1:300), donkey anti-goat IgG H&L-HRP (ab97110, 1:2000). Mouse monoclonal anti-ODC (sc-398116, 1:100 dilution) and goat anti-mouse IgG-HRP (sc-2005, 1:2500 dilution) antibodies were from Santa-Cruz Biotechnology, Heidelberg, Germany.
3
Immunoblot Analysis of Protein Expression
4
Protein Expression Analysis in Zebrafish Larvae
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Larvae aged 54 hpf were deyolked using Ginsburg Fish Ringer (NaCl, KCl, NaHCO3) solution. For each condition, 40 larvae were homogenized using 1x RIPA buffer (EMD Millipore RIPA lysis Buffer 10×) and protease inhibitor cocktail (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail). Lysate was recovered following centrifugation. For each sample, protein was quantified using a Bradford assay, and 35 μg at equal concentration were loaded on 12.5% SDS polyacrylamide gel. Separated proteins were transferred to a nitrocellulose membrane and blocked in 5% milk (TSBT) for 1 hr at room temperature. Membrane was incubated with myc primary antibody (monoclonal, Cell Signaling Technology (9b11), 1:1000) in blocking buffer at 4 °C overnight. Mouse secondary antibody (Jackson Immuno, 1:10 000) was applied for 1 hour at room temperature, and enhanced chemiluminescence was used for visualization. Following stripping using a mild stripping buffer (Glycine, SDS, Tween 20, pH 2.2), membrane was re-probed with Actin (C4 MPBio, 1: 10 000), and visualised using the same method as previously described.
5
Protein Expression Analysis by Western Blotting
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Western blotting was modified from our previous study [56 (link)]. Arsenic-treated M0/M1/M2 cells were incubated in RIPA lysis buffer 10× (20–188, Merck, Germany) with a cocktail of protease inhibitors for 2 h at 4 °C. The protein centration in the cell lysates was measured using the BCA protein quantification kit (Bio-Rad, Hercules, CA, USA). In total, 40 μg of proteins each sample was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the proteins in the polyacrylamide gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The nitrocellulose membranes were incubated with anti-GAPDH (GTX100118, GeneTex, Irvine, CA), anti-LC3 (Ab192890, Abcam, UK), anti-p62/SQSTM1 (ab91526, Abcam), anti-PINK1 (6946, Cell Signaling, Danvers, MA, USA), or anti-p-Parkin (36866, Cell Signaling) antibodies at room temperature for 12 h and then incubated with anti-Rabbit HRP-conjugated secondary antibodies (7074, Cell Signaling, Danvers, Massachusetts) or anti-Mouse HRP-conjugated secondary antibodies (7076, Cell Signaling, Danvers, Massachusetts) for 2 h at room temperature. After the antibody reactions, the western blotting bands were visualized by a chemiluminescence substrate kit (Pierce, Rockford, IL, USA).
6
Nanoparticle-Induced ER Stress Analysis
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The ER stress markers were identified
by Western blot analysis, using a Wes Simple Western Blot device (ProteinSimple).
Cells were seeded at 3 × 105 cells per well in a 6-well
plate and incubated for 2–3 days. Cells were then treated with
cubic ND-PEG-SPIONs (0.100 mg/mL Fe) for 24 h. After replacing particle-containing
media with fresh media, cells were treated with pulsed magnetic field
and then incubated for 3 h at 37 °C, 5% CO2. Cells
were abundantly washed with PBS, trypsinized, and centrifuged, and
then 100 μL of RIPA lysis buffer 10× (EMD Millipore; RIPA
containing protease inhibitor cocktail from cOmplete) was applied
to each well for 0.5 h. Then, 30 uL (out of 100 uL of lysate, 70 uL
was used for next step) of lysate diluted 3× using DPBS was used
for BCA analysis as described above to determine protein concentrations.
Using BCA data, 250 μg/mL of protein was denatured and loaded
in Wes multiwell plates following the manufacturer’s protocol.
The protein bands were detected with primary antibodies described
inTable S2 (Supporting Information) and
secondary Goat Anti-Rabbit HRP Conjugate (ready-to-use reagent, ProteinSimple).78 (link)
by Western blot analysis, using a Wes Simple Western Blot device (ProteinSimple).
Cells were seeded at 3 × 105 cells per well in a 6-well
plate and incubated for 2–3 days. Cells were then treated with
cubic ND-PEG-SPIONs (0.100 mg/mL Fe) for 24 h. After replacing particle-containing
media with fresh media, cells were treated with pulsed magnetic field
and then incubated for 3 h at 37 °C, 5% CO2. Cells
were abundantly washed with PBS, trypsinized, and centrifuged, and
then 100 μL of RIPA lysis buffer 10× (EMD Millipore; RIPA
containing protease inhibitor cocktail from cOmplete) was applied
to each well for 0.5 h. Then, 30 uL (out of 100 uL of lysate, 70 uL
was used for next step) of lysate diluted 3× using DPBS was used
for BCA analysis as described above to determine protein concentrations.
Using BCA data, 250 μg/mL of protein was denatured and loaded
in Wes multiwell plates following the manufacturer’s protocol.
The protein bands were detected with primary antibodies described
in
secondary Goat Anti-Rabbit HRP Conjugate (ready-to-use reagent, ProteinSimple).78 (link)
7
Matrigel-based Cell Culture and Transfection
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Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix containing purified human collagen I, laminin 1, vitronectin, and fibronectin was purchased from CORNING, NY, USA. DNA constructs and expression vectors were transfected into targeting cells by using Turbofect transfection reagent (Thermo-Fisher Scientific, Waltham, MA, USA). RIPA lysis buffer 10× was obtained from EMD Millipore (#20-188). Tablets of the cOmplete™ EDTA-free Protease Inhibitor Cocktail and the PhosSTOP™ Phosphatase inhibitor were purchased from Roche. The proteome profiler human phospho-kinase array kit was purchased from R&D Systems (Minneapolis, MN, USA) (catalog # ARY003B).
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