Western blots were performed using standard protocols: protein lysates were resolved in 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The primary antibodies and dilutions used were the following: rabbit polyclonal anti-NIS (1:500; 24324-1-AP, Proteintech, Rosemont, IL, USA); rabbit polyclonal anti-GFP (1:10,000; ab290, Abcam, Cambridge, UK); rabbit poly-clonal anti-ERK 1/2 antibody (1:1000; #4695, Cell Signaling, Danvers, MA, USA); mouse monoclonal anti-HA (1:10,000; 11 583 816 001, Roche, Mannheim, Germany); mouse monoclonal anti-phospho-ERK anti-body (1:1000; M8159, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal anti-PCNA (1:2000; NA03, Merck, Darmstadt, Germany); mouse monoclonal anti-RAC1 (1:1000; 05-389, Millipore, Burlington, MA, USA) and mouse monoclonal anti-alpha tubulin (1:300,000; T5168, Sigma-Aldrich, St. Louis, MO, USA). HRP-labeled anti-rabbit or anti-mouse IgG secondary antibodies (1:3000; Bio-Rad, Hercules, CA, USA) were used for chemiluminescence imaging.
M8159
Manufactured by Merck Group
Sourced in United States
The M8159 is a laboratory equipment product offered by the Merck Group. It serves as a general-purpose piece of lab equipment, designed to perform fundamental laboratory tasks. The core function of the M8159 is to provide a reliable and versatile tool for researchers and scientists working in various laboratory settings. No further details or interpretations about the intended use of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab290, by Abcam (1 mentions)
Anti phospho erk antibody, by Merck Group (1 mentions)
Hrp labeled anti rabbit or anti mouse igg secondary antibodies, by Bio-Rad (1 mentions)
T5168, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
M5670, by Merck Group (1 mentions)
Nb600 501, by Novus Biologicals (1 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
C9848, by Merck Group (1 mentions)
Bandpass filter cube, by Nikon (1 mentions)
C4731, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Biotin rna labeling kit, by Roche (1 mentions)
Gtx112141, by GeneTex (1 mentions)
A11031, by Thermo Fisher Scientific (1 mentions)
A11036, by Thermo Fisher Scientific (1 mentions)
Ab51098, by Abcam (1 mentions)
A11006, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
10 protocols using m8159
1
Western Blot Standardized Protocols
2
Western Blot Analysis of MAPK Signaling
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Cortical and hippocampal samples were homogenized in lysis buffer (50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1% Triton-X100, 1 mM DTT) supplemented with protease and phosphatase inhibitor cocktails (Roche) at 4 °C, and the lysates were subjected to SDS-PAGE analysis. Gels were transferred to nitrocellulose membranes and probed with the following antibodies: diphosphorylated Erk1/2 (1:1000; M8159, RRID: AB_477245, Sigma Aldrich), Erk1/2 (1:50,000; M5670, RRID: AB_477216, Sigma Aldrich), phospho-p38 MAPK (1:500; 4511S, RRID: AB_2139682, Cell Signaling Technology), p38 MAPK (1:1000; 9212S, RRID: AB_330713, Cell Signaling Technology), phospho-JNK1/2/3 (1:2000; MABS740; Merck Millipore), JNK1/23 (1:2000; 10023-1-AP, RRID:AB 2281669, Proteintech Group), occludin (1:1000; 40-4700, RRID: AB_2533468, Thermo Fisher Scientific), and β-actin (1:100,000, NB600-501, RRID: AB_10077656, Novus Biologicals). PageRuler Plus Prestained Protein Ladder (#26619, Thermo Scientific) was used as the molecular weight marker. Blots were quantified using Fiji-ImageJ software. The densitometry data were normalized either to the total form of the corresponding kinase or to β-actin.
3
Immunohistochemical analysis of retinal development
4
Quantifying MAPK Phosphorylation in Embryos
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M8159 (mouse monoclonal, Sigma-Aldrich M8159), an antibody for phosphorylated ERK1/2, was used to quantify MAPK (ERK1/2) phosphorylation after either T4 exposure. Embryos were exposed to 100 nM T4 for 90 min prior to fixation.
In a second experiment to localize TH binding, gastrulae were also stained with a custom 6a9 antibody (mouse monoclonal, kindly provided by the Ettensohn lab at Carnegie Mellon University) for primary mesenchyme cell membranes. This was performed in conjunction with the procedure for fluorescently labeled THs described below.
Embryos were fixed for whole mount immunohistochemistry in methanol at −20°C for 5 min before being transferred to PBS. After immunohistochemistry, larvae were imaged under fluorescent light with a Cy5 filter (Nikon Bandpass filter cube, excitation 620 nm, emission 700 nm). MAPK phosphorylation levels were quantified as mean fluorescence intensity of the embryo.
In a second experiment to localize TH binding, gastrulae were also stained with a custom 6a9 antibody (mouse monoclonal, kindly provided by the Ettensohn lab at Carnegie Mellon University) for primary mesenchyme cell membranes. This was performed in conjunction with the procedure for fluorescently labeled THs described below.
Embryos were fixed for whole mount immunohistochemistry in methanol at −20°C for 5 min before being transferred to PBS. After immunohistochemistry, larvae were imaged under fluorescent light with a Cy5 filter (Nikon Bandpass filter cube, excitation 620 nm, emission 700 nm). MAPK phosphorylation levels were quantified as mean fluorescence intensity of the embryo.
5
Hippocampal Cultures Protein Analysis
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14DIV hippocampal cultures were lysed in lysis buffer with 5% SDS and analyzed by Western blotting using mouse anti‐phospho ERK1/2 (1:1,000, M8159, Sigma‐Aldrich, Milan, Italy), rabbit anti‐calnexin (1:2,000, c4731, Sigma‐Aldrich, Milan, Italy), rabbit anti‐SHANK2 (1:2,000; 162202, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:10,000; UC Davis/NIH NeuroMab Facility, CA), rabbit anti‐SV2a (1:2,000; 119002, Synaptic Systems, Goettingen, Germany), guinea pig anti‐vGlut1 (1:1,000; 135304, Synaptic Systems, Goettingen, Germany), mouse anti‐SNAP‐25 (1:100,000; SMI81 Sternberger Monoclonals, Baltimore MD), rabbit anti‐GAPDH (1:4,000, 247002, Synaptic Systems, Goettingen, Germany). Membranes were washed and incubated for 1 h at room temperature with the secondary antibody anti‐mouse or anti‐rabbit HRP conjugated (Thermo Fisher, Waltham, MA, USA). Blots were scanned using a ChemiDoc‐MP system (Bio‐Rad, Segrate, Italy). Optical densities were measured using the Fiji software (NIH, Bethesda, Maryland, USA) with local background subtracted as described Fossati et al (2015 ). For each sample, calnexin was used as a loading control.
6
Antibody and in situ Hybridization Protocols
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Antibody staining and in situ hybridization was conducted essentially as described previously [35 , 43 ]. The following antibodies were used: guinea pig anti-Al (1:1000) [44 ], goat anti-Dll (1:2, dF-20, Santa Cruz Biotechnology, Dallas, USA; pre-adsorbed with silkworm larval epidermal powder), rat anti-Pros (1:5, a gift from F. Matsuzaki), mouse anti-Notch (1:40, C17.9C6, Developmental Studies Hybridoma Bank, Iowa City, USA), mouse anti-Arm (1:40, N2 7A1, Developmental Studies Hybridoma Bank, Iowa City, USA), mouse anti-diphospho ERKI/II (1:250, M8159, Sigma-Aldrich, St. Louis, USA), and fluorophore (Alexa Fluor 488, 555, 647)-conjugated secondary antibodies (1:100, Thermo Fisher Scientific, Waltham, USA or Jackson ImmunoResearch, West Grove,USA). Riboprobes for in situ hybridization of wg and rho in B. mori were generated with DIG or Biotin RNA labeling kit (Roche, Basel, Switzerland) using cDNA as a template. Primers used are listed in Additional file 5 : Table S1. The detailed procedures are described in Additional file 6 .
7
Molecular Signaling Modulation in Inflammatory Response
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Cells (5 × 105 cells) were seeded in 6-cm cell culture dishes for 24 h. Cells were then treated with LPS (100 ng/mL) or SARS-CoV-2-E (1 μg/mL), followed by GMI (0, 0.3 and 0.6 μM) for 24 h. After treatment, cells were harvested and rinsed with cold PBS containing 1 % Na3VO4 and lysed using a specific lysis buffer with protease inhibitor [27] . Cell lysates were collected by centrifugation at 13,000 ×g for 10 min at 4 °C. Cell lysates (30 μg) were separated on 10 % SDS-PAGE and specific molecules were detected using a western blot analysis, using the method of a previous study [28] (link). Antibodies against COX-2 (SC-1747) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies against iNOS (GTX74171) and tubulin (GTX112141) were purchased from GeneTex (Hsinchu, Taiwan). Antibodies against phosphorylated JNK (J4750), ERK1/2 (M8159) and P38 (M8177) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
8
Tissue Immunostaining with Antibodies
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Tissue was dissected in phosphate-buffered saline (PBS), fixed in 4% PFA/PBS for 30′ on ice, washed in PBS-T, incubated with primary antibody in PAXDG (PBS containing 1% BSA, 0.3% Triton X-100, 0.3% deoxycholate and 5% goat serum), followed by washing and incubation with secondary antibody in PAXDG and subsequent mounting in Vectashield with DAPI (Vector Laboratories). Primary antibodies used were: rat anti-myosin (ab51098; Abcam; 1:100) and mouse anti-pERK (M-8159; Sigma; 1:50). Polyclonal rabbit anti-CG1139 (1:200) was purified by New England Peptide Inc., MA, USA. An amino acid sequence 26–40 was selected as the epitope. The following primary antibodies were kindly gifted to us: rat anti-bnl (M. Krasnow; 1:50), guinea pig anti-Path (J. Parrish; 1:200) and rabbit anti-phospho-Drosophila S6 (pS6) (A. Teleman; 1:500). Secondary antibodies used were: Alexa Fluor 488 and 568 conjugated anti-rat antibody (A-11006 and A-11077; Thermo Fisher Scientific; 1:200), Alexa Fluor 568 conjugated anti-mouse antibody (A-11031; Thermo Fisher Scientific; 1:200), Alexa Fluor 568 conjugated anti-rabbit antibody (A-11036; Thermo Fisher Scientific; 1:200) and Alexa-568 conjugated anti-guinea pig antibody (A-11075; Thermo Fisher Scientific, 1:200). Rhodamine phalloidin (R415; Invitrogen; 1:500) was used to visualise F-actin.
9
Muscle, Neural Crest, and MAPK Staining
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Specimens for antibody staining were fixed in Dent’s fixative. Muscles were labeled with 12/101 antibody (AB531892; Developmental Studies Hybridoma Bank), neural crest cells were labeled with Sox9 antibody (AB5535; Merck Millipore), basal lamina was labeled with anti-fibronectin (A0245; DAKO) and MAPK activity was assessed using anti-activated MAP kinase antibody (M8159; Sigma). Primary antibodies were detected by Alexa Fluor 488 and 594 (Invitrogen, Thermo Fisher Scientific Inc.). Visualisation of nerve fibres was performed using anti-acetylated tubulin antibody (T6793; Sigma) and EnzMet Enzyme Metallography kit (Nanoprobes).
10
Antibody-Based Protein Detection Protocols
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Antibodies against the following proteins were used for this study:
Actin (C4, kMillipore), ATP synthase beta (ab14730 Abcam), Cytochrome c oxidase subunit 1 (ab14705 Abcam), dpMPK (M8159, Sigma), GFP (Wako), HIM-4 (gift from Bruce Vogel) [117 (link)], NPP-9 [36 (link)], PGL-1 (gift from Susan Strome) [37 (link)], alpha-Tubulin (YOL3/4 Abcam), UNC-60/cofilin (gift from Shoichiro Ono) [61 (link)].
Actin (C4, kMillipore), ATP synthase beta (ab14730 Abcam), Cytochrome c oxidase subunit 1 (ab14705 Abcam), dpMPK (M8159, Sigma), GFP (Wako), HIM-4 (gift from Bruce Vogel) [117 (link)], NPP-9 [36 (link)], PGL-1 (gift from Susan Strome) [37 (link)], alpha-Tubulin (YOL3/4 Abcam), UNC-60/cofilin (gift from Shoichiro Ono) [61 (link)].
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