Cells were lysed in RIPA buffer complemented with Set I and Set II phosphatase inhibitors at 1× (Calbiochem), and protease inhibitors at 1× (Roche). Whole cell lysate concentration was determined with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Proteins were resolved on SDS-PAGE gels and electrotransferred to nitrocellulose membranes, 0.2 µm (Bio-Rad). Primary antibodies pS6S235/236, S6, pAKTS473, AKT, p4E-BP1T37/46, 4E-BP1, Cleaved PARP, pAktT308, p62, Rictor, Raptor, HIF-1α, HIF-2α, pERK1/2T202/Y204 (mouse), ERK, p-p90RSKS380, RSK1/2/3, pBADS112, pBADS136, pEGFRY1068, cleaved-caspase3 were from Cell Signaling Technologies. VHL (Santa Cruz #FL-181). mTOR primary antibody was from Millipore. Primary antibody dilutions were to manufactures' specifications (See Table S1 ). Tubulin (Sigma #T5168), KU-80 (GeneTex #GTX70485) and Actin-HRP (Santa Cruz #C-11) primary antibodies served as loading controls (LC) where noted. Secondary anti-Rabbit and ant-mouse antibodies were from (Fisher) and diluted in 5% milk, 1× TBS-T solution. ECL Western Blotting Detection reagents (GE Healthcare) were used for developing blots onto autoradiography film. For difficult to detect proteins SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used in combination with ECL.
Actin hrp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Actin-HRP is a conjugate of actin, a cytoskeletal protein, and horseradish peroxidase (HRP), an enzyme used as a detection label. It is designed for use in various research applications that require the identification and visualization of actin.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Goat anti mouse alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Gata3, by R&D Systems (2 mentions)
Sema3b, by Santa Cruz Biotechnology (2 mentions)
Phoshop limk1 thr508 limk2 thr505, by Cell Signaling Technology (2 mentions)
Limk2 8c11, by Cell Signaling Technology (2 mentions)
Ly294002, by Merck Group (2 mentions)
P gsk3β, by Cell Signaling Technology (2 mentions)
Erk1 2, by Santa Cruz Biotechnology (2 mentions)
P erk1 2, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Hif 2α, by Cell Signaling Technology (1 mentions)
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
17 protocols using actin hrp
1
Western Blot Analysis of Signaling Proteins
2
Biochemical Analysis of Key Signaling Pathways
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Cells were pelleted by centrifugation, washed with ice-cold PBS and lysed on ice for 20 min in modified NP-40 buffer (10mM/1mM KPO4/EDTA pH 7.05, 5mM EGTA pH 7.2, 10mM MgCl2, 50mM glycerophosphate pH 7.2, 0.5% NP-40, 0.1% Brij-35) supplemented with protease inhibitor mixture (Roche), phosphatase inhibitor mixture (Roche), 1mM DTT, 1mM Va3VO4 and 1mM PMSF. Protein concentrations of whole-cell lysates were determined by BCA protein assays (Pierce), and equivalent protein amounts (20-60 μg) were electrophoresed through 4-12% Bis-Tris acrylamide gels. Proteins were transferred to 0.45 μM nitrocellulose membranes (Invitrogen) and blocked with 5% milk. Antibodies detecting pAKT(Ser473)XP, pAKT(Thr 308), AKT, pPRAS40(Thr246), PRAS40, p110α, p110β, p110γ, p85, Syk, pSyk(Tyr525/526), pZap-70(Tyr319)/Syk(Tyr352), pSrc(Tyr416), pCD19(Tyr531), CD19, pPLCγ2(Tyr759), pCD79A(Tyr182), pFoxO1(Thr24)/FoxO3a(Thr32)/FoxO4(Thr28), CD79, p-tyrosine were purchased from Cell Signaling. CD19, Syk, p110δ and actin-HRP were purchased from Santa Cruz. BCAP was purchased from R&D. Anti-rabbit IgG and anti-mouse IgG (peroxidase-conjugated) were purchased from GE Healthcare Life Sciences. Light chain-specific anti-mouse IgG, Anti-rabbit IgG and anti-goat IgG (peroxidase-conjugated) were purchased from Jackson ImmunoResearch.
3
Immunoblotting of Chromatin Remodeling Proteins
4
Quantitative Analysis of OPA1 Protein
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Lungs were homogenized, and protein concentrations were measured with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). An equal amount of protein (25 mg) was separated in a SDS gel. Appropriate primary antibodies were purchased from Actin-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and OPA1 (BD Biosciences, San Jose, CA, USA). Secondary antibodies conjugated with horseradish peroxidase (Life Technologies, Grand Island, NY, USA) were applied according to the manufacturer’s protocols. Quantification of the proteins were obtained with the CS analyzer 3.0 program (ATTO, Tokyo, Japan).
The OPA1 protein(50ug) were separated on SDS-PAGE and transferred to the nitrocellulose membranes using a wet transfer method on 12V for 1H. The membranes were pre-blocked for 1H in Tris-buffered saline (TBS) + Tween 20 solution containing 5% skimmed milk overnight at 4°C with agitation. Subsequently, solutions were incubated with either base-1 IgG (rabbit polyclonal antibody) or GAPDH IgG (rabbit polyclonal antivbody) overnight at 4°C. After washing by TBS+Tween 20 for 15min, goat anti-rabbit IgG-HRP was treated and incubated for 1H with agitation. The bands of interest were detected with CCD cameral gel documentation system(chemidoc; Bio-rad, USA).
The OPA1 protein(50ug) were separated on SDS-PAGE and transferred to the nitrocellulose membranes using a wet transfer method on 12V for 1H. The membranes were pre-blocked for 1H in Tris-buffered saline (TBS) + Tween 20 solution containing 5% skimmed milk overnight at 4°C with agitation. Subsequently, solutions were incubated with either base-1 IgG (rabbit polyclonal antibody) or GAPDH IgG (rabbit polyclonal antivbody) overnight at 4°C. After washing by TBS+Tween 20 for 15min, goat anti-rabbit IgG-HRP was treated and incubated for 1H with agitation. The bands of interest were detected with CCD cameral gel documentation system(chemidoc; Bio-rad, USA).
5
Embryonic Cortex Protein Extraction and Western Blot
6
Quantifying Actin-Mediated Signaling Pathways
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Actin-HRP (cs-47778, Santa Cruz Biotechnology), Alexa Fluor 546 goat anti-mouse (A-11008, ThermoFisher Scientific), Alexa Fluor 488 goat anti-rabbit (A-11030, ThermoFisher Scientific), GATA3 (AF2605, R&D Systems, Ki67 (ab15580, Abcam), Sema3B (sc-21204-R, Santa Cruz Biotechnology). Phoshop-LIMK1 (Thr508)/LIMK2 (Thr505) (3841T, Cell Signaling Technology). LIMK1 (VMA00361KT, Bio-Rad Laboratories Inc.). LIMK2 (8C11) (3845, Cell Signaling Technology).
7
Quantifying Actin-Mediated Signaling Pathways
8
Lysosomal Marker and Autophagy Protein Detection
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Primary antibodies against LAMP1, LAMP2, Cathepsin D, Cathepsin L, p62, galectin-3 and secondary goat anti-mouse IgG (Alexa Fluor 647) were purchased from Abcam (Cambridge, UK). LC3A/B was from Cell Signaling (Bioke, Leiden, The Netherlands), actin-HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CD86-Alexa700 and HLA-DR-PeCy5 from BD Biosciences (Erembodegem, Belgium) and CD80-BV650, CD14-FITC and CD163-Alexa647 were bought from Biolegend (ITK diagnostics, Uithoorn, The Netherlands). NDP52 (CALCOCO2), secondary goat anti-rabbit IgG (Alexa Fluor 647) and HRP-conjugated antibodies reactive with mouse and rabbit were purchased from Thermo Fisher Scientific (Merelbeke, Belgium).
9
Western Blot Antibody Reagents
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Unless otherwise stated, all reagents and media were purchased from Fisher Scientific (Pittsburgh, PA.) Dox and LY294002 were purchased from Sigma-Aldrich, St. Louis, MO. N-benzyladriamycin-14-valerate (AD198) was a kind gift from Dr. Leonard Lothstein (The University of Tennessee, Health Science Center in Memphis) [21 (link)]. The following antibodies were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA): Actin-HRP, p-ERK1/2, ERK1/2, AKT and p38. The following antibodies were purchased from Cell Signaling (Boston, MA): PARP, p-AKT (Serine 473 and Threonine 308), p-GSK3β and p-p38.
10
Whole-cell Protein Extraction and Western Blot
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Whole-cell extracts were prepared using NP-40 lysis buffer (20 mM Tris–HCl, pH 8.8, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 12 mM Na-deoxycholate). Cell homogenates were incubated for 30 min on ice followed by a clean-up centrifugation at 20,000 × g for 10 min at 4°C. Protein concentration was determined with Pierce BCA Protein Assay Reagent (Thermo Scientific) using bovine serum albumin as a standard. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose (Bio-Rad) membrane. The membrane was blocked with StartingBlock Blocking Buffer (Thermo Scientific) for 1 h followed by incubation with primary antibody: PARP-1 (1:1,000; Cell Signaling, Cat#9532), PAX7 (1:1,000; Abcam, Cat#ab34360), myogenin (1:1,000; Abcam, ab124800), PCNA (1:1,000; Cell Signaling, Cat#2586), Histone H3 (1:1,000; Cell Signaling, Cat#12648P), ATP synthase (subunit alpha) (1:1000, Life Technologies, Cat#459240/G0531), actin-HRP (1:5,000; Santa Cruz Biotechnology, Cat#sc-1616 HRP); followed by incubation with anti-mouse or anti-rabbit secondary antibodies (Cell Signaling). The membrane was developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and visualized in a GeneBox Detection System (Syngene).
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