Hans' Electroacupunture apparatus (Catalog No. hans-200a/100b, Beijing, China)，is a kind of small portable electroacupuncture instrument approved for using for treating patients, or used as a tool in research studies. The primary antibodies, including the anti-MYOD antibody (ab203383), anti-PAX7 antibody (ab187339), and anti-p38 MAPK (mitogen-activated protein kinases) (sc-166182), were purchased from Santa Cruz Biotechnology, Inc., Company (Oregon, USA) and diluted at 1 : 1000 when used. The secondary antibody, Goat Anti-Rabbit IgG H&L (FITC) (ab6717), was purchased from Abcam Company (Cambridge, MA 02139-1517, USA).
Sc 166182
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-166182 is a laboratory equipment product offered by Santa Cruz Biotechnology. The core function of this product is to facilitate scientific research and experiments, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l fitc ab6717, by Abcam (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Doxorubicin dox, by Merck Group (1 mentions)
Mab374, by Cell Signaling Technology (1 mentions)
Phospho p38 tyr182, by Santa Cruz Biotechnology (1 mentions)
Vegfa, by Proteintech (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Sch772984, by Topscience (1 mentions)
Sc 7972, by Santa Cruz Biotechnology (1 mentions)
Sc 32282, by Santa Cruz Biotechnology (1 mentions)
Gapdh antibody 60004 1 ig, by Proteintech (1 mentions)
Sc 271966, by Santa Cruz Biotechnology (1 mentions)
Sc 56053, by Santa Cruz Biotechnology (1 mentions)
11 protocols using sc 166182
1
Electroacupuncture Instrument for Muscle Research
2
DEHP and Doxorubicin Signaling Pathway Analysis
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DEHP (Sigma-Aldrich, 36735, Saint Louis, MO, USA) was reconstituted in DMSO (Sigma-Aldrich, Saint Louis, MO, USA). Doxorubicin (Dox) (Sigma-Aldrich, D1515, Saint Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich, Saint Louis, MO, USA). Primary antibodies for proteins: Endoglin/CD105 (71 kDa, Proteintech, 10862-1-AP, Rosemont, IL, USA), MAPK (ERK1/2) (44 kDa, Cell Signaling, 137F5, Danvers, MA, USA), phospho-MAPK (ERK1/2) (Thr202/Tyr204) (44 kDa, Cell Signaling, 9101 s, Danvers, MA, USA), p38 (38 kDa, Proteintech, 14064-1-AP, Rosemont, IL, USA), phospho-p38 (Tyr182) (38 kDa, Santa Cruz, SC-166182, Santa Cruz, CA, USA), GAPDH (35.5 kDa, EMD Millipore, MAB374, Burlington, MA, USA), Smad2/3 (52, 60 kDa, Cell Signaling, 8685, Danvers, MA, USA), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (52, 60 kDa, Cell Signaling, 8828, Danvers, MA, USA), β actin (43 kDa, Santa Cruz, SC-47778, Santa Cruz, CA, USA), and VEGFA (43 kDa, Proteintech, 66828-1-IG, Rosemont, IL, USA). Secondary antibodies: Goat anti-rabbit IgG (Alexa Fluor 594, Invitrogen, A11012, Waltham, MA, USA) was used as the secondary antibody.
3
Assessing α-Synuclein Aggregation Mechanisms
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SCH772984 was purchased from Topscience Co., Ltd.; N‐acetyl‐L‐cystine (NAC, A7520), Hoechst 33342 (B2261), PI (P4170), and dihydroethidium (DHE, 309800) were purchased from Sigma Chemical Co. Dulbecco's modified Eagle's medium (DMEM), Trypsin–EDTA, and penicillin–streptomycin were purchased from Thermo Fisher Scientific Inc. Fetal bovine serum (FBS, #164210) was obtained from Procell Life Science & Technology Co., Ltd. YFP‐CL1 (11950), EGFP‐α‐synuclein‐WT (40822), EGFP‐α‐synuclein‐A53T (40823), and pHM6‐α‐synuclein‐A53T (40825) plasmids were purchased from Addgene (Addgene Inc.). EGFP‐N1 (6085/1/1), EGFP‐α‐synuclein‐A30P (PPL01823‐2b), and EGFP‐α‐synuclein‐E46K (PPL01823‐2c) plasmids were purchased from Public Protein/Plasmid Library (PPL). The commercially available antibodies used in this study include Bax (14,796S), Bcl‐2 (3498S), ERK (4695S), JNK (4668S), α‐actinin (3134S), and p‐JNK (4668S) from Cell Signaling Technology Inc. (CST); p38 MAPK (sc‐7972), p‐p38 MAPK (sc‐166,182), Caspase‐3 (sc‐390,394), and β‐actin (sc‐47,778) from Santa Cruz Biotechnology Inc; GAPDH (60004‐1‐Ig), Caspase‐9 (10380‐1‐AP), and PARP1 (13371‐I‐AP) from Proteintech Group, Inc; p‐ERK (ap0974) from ABclonal Biotechnology Co., Ltd.; and GFP (MO49‐3) from MBL International. The specificity of all antibodies was verified by the suppliers.
4
Protodioscin-Induced Apoptosis and Mitophagy
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Protodioscin (PD; CFN99517) with 99% purity was purchased from ChemFaces (Wuhan, Hubei, China). The MTT, DAPI (4′, 6-diamidino-2-phenylindole), and Hoechst 33258 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against cleaved caspase-9 (9508S) and cleaved PARP (9542S) were purchased from Cell Signaling Technology (Danvers, MA, USA). LC3 antibody (NB100-2220) was purchased from Novus Biologicals (Centennial, CO, USA). Antibodies against cleaved caspase-caspase-3 (SC-56053), PINK1 (SC-518052), Parkin (SC-32282), NIX (SC-166332), BNIP3 (SC-56167), total p38MAPK (SC-7972), phosphoryl p38MAPK (SC-166182), total Akt (SC-56878), phosphoryl Akt (SC-271966), LC3 siRNA (SC-43390), NIX siRNA (SC-37453), and horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). GAPDH (60004-1-Ig) antibody was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). The p38 siRNA (MAPK14-Homo-760) was purchased from AllBio Company (Taichung, Taiwan).
5
AMPK Signaling Pathway Analysis
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Antibodies against ICAM-1 (SC-107), p-AMPK (SC-33524), AMPK (SC-25792), p-p38 (SC-166182), p38 (SC-271120) and β-actin (SC-47778) were all bought from Santa Cruz (Santa Cruz, CA, USA). All ON-TARGETplus siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Cell culture supplements were purchased from Invitrogen (Carlsbad, CA, USA). A Dual-Luciferase® Reporter Assay System was bought from Promega (Madison, WI, USA). qPCR primers and probes, as well as the Taqman® one-step PCR Master Mix, were supplied by Applied Biosystems (Foster City, CA, USA). All other chemicals not mentioned above were supplied by Sigma-Aldrich (St. Louis, MO, USA).
6
Immunofluorescence Analysis of NF-κB and MAPK Signaling
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Following the culture and treatments, cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) and blocked with 5% normal goat serum (MultiSciences, Hangzhou, China) in 0.3% Triton-X-PBS and then immunostained for p65 (1:100, sc-71675, Santa Cruz, USA), p-p65 (1:100, sc-166748, Santa Cruz, USA), p-p38 (1:100, sc-166182, Santa Cruz, USA), and Iba-1 (1:100, sc-32725, Santa Cruz, USA). Alexa Fluor 594-conjugated goat antimouse IgG (1:200, ab150116, Abcam, Cambridge, England) was used as secondary antibody. DAPI (Biomol, Hamburg, Germany) was used for counterstaining [54 (link)]. Fluorescence images were captured by a fluorescence microscope system (OLYMPUS IX73, Olympus, Japan). The images were analyzed with software Image J (Image J Version 1.52e, National Institutes of Health, Bethesda, MD, USA).
7
Chemiluminescent ROS Detection Assay
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The chemiluminescent probe, Bis-N-methylacridinium nitrate (lucigenin, catalogue no. 2315-97-1); the reagents L-NAME (N5751), Rotenone (R8875), Oxypurinol (O6881), Diphenyleneiodonium chloride (DPI, D2926), Triton X-100 (T8787) and Tiron (D7389); fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (Lectin, L4895) and; fluorochrome-conjugated anti-goat (C2821) or anti-rabbit (C2306) secondary antibodies were purchased from Sigma-Aldrich, Dorset, UK and gp91 ds-tat (DS-TAT, AS-63818) was bought from AnaSpec, Fremont, USA. NADPH (AC328742500) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. Dihydroethidium (DHE, D11347) was from Invitrogen, Paisley, UK. Polyclonal antibodies targeting Nox2 (sc-20782), Nox4 (sc-30141), phospho-Akt1/2/3 (sc-16646-R), total Akt1/2/3 (sc-8312), total ERK1/2 (sc-292838), total-p38MAPK (sc-535), α-tubulin (sc-5546) and the monoclonal antibody targeting phospho-p38MAPK (sc-166182) were from Santa Cruz Biotechnology, Dallas, TX, USA. Phospho-ERK1/2 (#9101) was purchased from Cell Signaling Technology, Danvers, MA, USA.
8
Immunohistochemistry Analysis of Rib Fracture
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For immunohistochemistry, dehydrated sections of rib fracture were treated with 0.3% H2O2 in phosphate-buffered saline (PBS; pH 7.4) for 30 min at room temperature to inactivate endogenous peroxidase. Sections were pretreated with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature, followed by incubation with primary antibodies against IL-1β (1:100, ab9722, Abcam, Cambridge, MA, USA), RARγ (1:100, #8965S, Cell Signaling Technology, Danvers, MA, USA), MMP-13 (1:100, ab39012, Abcam), CCN2 (1:100, ab6992, Abcam), α-SMA (1:1000, M0851, DakoCytomation, Glostrup, Denmark), and p-p38 (1:100, sc166182, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Sections were reacted with Histofine Simple Stain rat MAX-PO (Multi; Nichirei, Tokyo) for 1 h at room temperature. Color was developed with the use of a liquid diaminobenzidine substrate-chromogen system (Dako, Carpinteria, CA, USA). Immunostained sections were then counterstained with methylene green.
9
Western Blot Analysis of Microglia
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Western blot assay was carried out as the previously described methods (Roman et al., 2020 (link)). Primary microglia and mouse microglia BV2 cells with different treatments were collected, and the total proteins were isolated using RIPA buffer (Gibco, United States). Then, the different protein samples were loaded into the lane of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by the transfer into polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, United States). Next, the membranes were placed in 5% skim milk and blocked at RT for 1 h and then incubated with the specific primary antibodies at 4°C overnight. The antibodies were: anti-CD86 (ab243887, 1:1000, Abcam), anti-CD206 (ab252921, 1:1000, Abcam), anti-p-JNK (sc-6254, 1:500, Santa Cruz Biotechnology), anti-JNK(sc-7345, 1:500, Santa Cruz Biotechnology), anti-p-p38 (sc-166182, 1:500, Santa Cruz Biotechnology), anti-p38 (ab170099, 1:2000, Abcam), anti-pERK (sc-7383, 1:500, Santa Cruz Biotechnology), anti-ERK (ab32537, 1:1000, Abcam) and β-actin (ab6276, 1:5000, Abcam). The samples were further incubated with the secondary antibody (ab205718, 1:2000, Abcam) at 37°C for nearly 1 h. Ultimately, the enhanced chemiluminescence reagents (Millipore, Bedford, MA) and ImageJ were applied to analyze protein bands.
10
Western Blot Analysis of Phospho-MAPK Signaling
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Protein expression was measured by Western blot as previously described (Siti et al., 2021 (link)). Anti-phospho-ERK1/2 rabbit polyclonal (1:1,000) (#4377), anti-phospho-JNK1/2 rabbit monoclonal (1:1,000) (#4668), and anti-phospho-p38 mouse monoclonal (1:500) (sc-166182; Santa Cruz Biotechnology, Dallas, TX, United States) were the primary antibodies used in this study. β-Actin mouse monoclonal antibodies (1:500) (sc-47778; Santa Cruz Biotechnology, Dallas, TX, United States) served as the loading control. HRP-conjugated IgG anti-mouse (1:2000) (sc-516102; Santa Cruz Biotechnology, Dallas, TX, United States) was used as the secondary antibody. Blots were visualized on a gel doc system and analyzed with ImageJ software (U. S. National Institutes of Health, Bethesda, MD, United States). A minimum of three biological replicates was performed in triplicate (n = 3).
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