Rabbit polyclone anti-CHOP, cytochrome c (Cyto C), ATF6, PERK, GRP78, ARF4, Bax, Bak, caspase3, cleaved caspase 3, caspase 8, caspase 9, IRE1, DR4, DR5, P-JNK, Bcl-2, Fas, β-Actin, Gapdh, and Tubullin were obtained from Affinity Biosciences (Shanghai, China). Mouse monoclonal anti-Lewis a (Lea), Lewis b (Leb), and Lewis y (Ley) were purchased from Abcam (Cambridgeshire, UK) [74 (link)]. Mouse monoclonal anti-SLex was purchased from BD Biosciences (San Diego, CA, USA) [75 (link)]. FITC-conjugated anti-mouse/rabbit IgG was purchased from Affinity Biosciences (Shanghai, China). Goat anti-rabbit IgG-HRP was obtained from Life Science. E-selectin and P-selectin were purchased from R&D System (Minneapolis, MN, USA). Lectins were purchased from Vector Laboratories (Burlingame, CA, USA). Cells were cultured with cell growth medium (Gibco, Shanghai, China) containing fetal bovine serum (FBS) (ABW, Shanghai, China) and penicillin/streptomycin (Gibco, Shanghai, China).
P erk
Manufactured by Affinity Biosciences
Sourced in United States, China
P-ERK is a product that measures the phosphorylation of extracellular signal-regulated kinase (ERK), a key signaling protein involved in cellular processes. It provides quantitative data on the activation state of ERK without further interpretation.
Automatically generated - may contain errors
Lab products found in correlation
P jnk, by Affinity Biosciences (5 mentions)
Gapdh, by Affinity Biosciences (5 mentions)
β actin, by Affinity Biosciences (2 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (2 mentions)
Caspase 3, by Affinity Biosciences (2 mentions)
Bcl 2, by Affinity Biosciences (2 mentions)
P akt, by Affinity Biosciences (2 mentions)
Caspase 9, by Affinity Biosciences (1 mentions)
Lectin, by Vector Laboratories (1 mentions)
Grp78, by Affinity Biosciences (1 mentions)
Caspase8, by Affinity Biosciences (1 mentions)
Cleaved caspase 3, by Affinity Biosciences (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
β actin, by ABclonal (1 mentions)
Lipopolysaccharide lps, by Solarbio (1 mentions)
Bca protein analysis kit, by Meilun (1 mentions)
C myc, by ZenBio (1 mentions)
Cyclin d1, by ZenBio (1 mentions)
14 protocols using p erk
1
Antibody Procurement for Cell Signaling
2
Western Blot Analysis of Protein Signaling
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Total proteins were isolated from mouse mammary gland and neutrophils with RIPA lysis buffe (Solarbio, Beijing, China) with added 1 mM phenylmethylsulfonyl fluoride (PMSF, Solarbio, Beijing, China). The supernatants were collected by centrifuging at 12,000 rpm for 10 min at 4°C. Protein concentration was measured by bicinchoninic acid assay (BCA) (Beyotime, Nantong, China). About 30 μg protein lysates were separated on polyacrylamide gel by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% bovine serum albumin diluted in Tris buffered saline with Tween-20 (TBST) for 2 h at room temperature and hybridized overnight with primary antibody (1:1000) at 4°C. Before and after incubation with the HRP-linked anti-rabbit IgG (1:10000, CST, MA, USA) at room temperature for 2 h, the membranes were washed 3 times with TBST. The signals were detected by an ECL Western blot analysis system (Tanon, Shanghai, China). Analysis of bands was quantifed with ImageJ software (NIH, USA). The primary antibodies were listed as follows: β-actin (ABclonal, Wuhan, China), TAK1, p-TAK1 (CST, MA, USA), p38, p-p38, ERK, p-ERK, JNK, p-JNK, p47phox, p-p47phox and TLR2 (Affinity, OH, USA).
3
Protein Expression Analysis in Wound Healing
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After incubation with PBS, OA-RD17 (1 nM), lipopolysaccharide (LPS, 1 μg/mL) (Solarbio, China), specific TLR4 inhibitor (1 μg/mL), miR-632 mimic (50 nM), or miR-632 inhibitor (100 nM) for 24 h, respectively, cell lysates (RIPA: PMSF: phosphatase inhibitor = 100:1:1; RIPA and PMSF, Meilun Biotechnology, Dalian, China; phosphatase inhibitor, Roche, Shanghai, China) were used to extract total protein in keratinocytes and macrophages. Moreover, cell lysates were also used to extract total protein in wound tissues from SD rats. The extracted proteins were quantified using the Bradford method (BCA protein analysis kit, Meilun, Dalian, China). The protein samples were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electro-imprinted on polyvinylidene fluoride membranes, and recorded and analyzed quantitatively using the Bole exposure software system. Primary antibodies, including GAPDH, Lamin B1, P38, P-P38, ERK, P-ERK, JNK, P-JNK, IκB, P-IκB, P65, P-P65 (Affinity, China), GSK3β, β-catenin, Cyclin D1, c-MYC, and Vimentin (ZEN BIO, China) were used following the provided instructions.
4
Immunofluorescence Staining of Lung Tissue
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Immunofluorescence (IF) was performed manually according to Camolotto et al. [42 (link)]. Briefly, lung tissues were fixed overnight in 4% paraformaldehyde and washed in PBS followed by serial dehydration with ethanol prior to embedding. Tissues were sliced into 6 μm thick sections, deparaffinized, rehydrated, and boiled for 22 min with Antigen Unmasking Solution (G1202, Servicebio, Wuhan, China). Slides were subsequently blocked in blocking buffer (3% BSA) for 1 h, incubated with primary antibodies at 4 °C overnight, and washed with PBS. FITC, Alexa-488 or Cy3-conjugated secondary antibodies were used to visualize the protein of interest via fluorescence. The following primary antibodies were used: PCNA (60097-1-Ig, Proteintech, Wuhan, China), SP-C (10774-1-AP, Proteintech, Wuhan, China), AQP5 (sc-514022, santa cruze, Dallas, USA), FGF10 (863308, ZENBIO, Chengdu, China), p-ERK (AF1015, Affinity, Jiangsu, China), ETV4 (10684-1-AP, Proteintech, Wuhan, China).
5
Inflammatory Bowel Disease Protocol
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Dextran Sulfate Sodium Salt (DSS) (Cat#9011-18-1) was purchased from MP Biomedicals (Irvine, CA). Specific antibodies including ZO-1 (1:1000; #AF5145; RRID:AB_2837631), Occludin (1:1000; #DF7504; RRID:AB_2841004), Claudin-3 (1:1000; #AF0129; #RRID:AB_2833313), p65 (1:1000; #BF8005:AB_2846809), p-p65 (1:1000; AB_2834435:AF2006), IKBα (1:1000; #AF5002:AB_2834792), p-IKBα (1:1000; #AB_2834433:AF2002), ERK (1:1000; #AB_2833336:AF0155), p-ERK (1:1000; #AB_2834432:AF1015), JNK (1:1000; #AF6318:AB_2835177), p-JNK (1:1000; #AF3318:AB_2834737), p38 (1:1000; #BF8015:Q16539), p-p38 (1:1000; #AF4001:AB_2835330) and GAPDH (1:1000; #AF7021; AB_2839421) were purchased from Affinity Biosciences (OH, USA). Tumor necrosis factor (TNF)-α (Cat# 430915), interleukin (IL)-1β (Cat# 432615), and interleukin (IL)-6 (Cat# 431307) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, California, USA). Myeloperoxidas (MPO) (A044-1-1), Superoxide Dismutase (T-SOD) (A001-3-2), Malondialdehyde (MDA) (A003-1-2), Glutathione Peroxidase (GSH-PX) (A005-1-2), and Catalase (CAT) (A007-1-1) assay kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
6
Signaling Pathway Antibody Protocol
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Lactate was obtained from Sigma (St. Louis, MO, USA), Antibody against DRP1(#8570), p-DRP1S616(#3455), p-IκB-alpha(#2859S), p-P65(#3033S) and P65(#8242) were purchased from Cell Signaling Technology (Boston, MO, USA). Antibody against fibronectin (#15,613–1-AP) and COL1A1(67,288–1-Ig) were purchased from Proteintech (Wuhan, China). Antibody against IκB(#AF5002), IKK(#AF6014), p-IKK(AF3013), ERK(#AF0155), p-ERK(#AF1015), β-Actin(#AF7018) and GAPDH(#AF7021) were purchased from Affinity (Changzhou, China).
7
Evaluation of Anti-Inflammatory Effects
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BA (purity over 90%) was purchased from Nanjing Zelang Biotechnology Co., Ltd. (Nanjing, China). The BA standard agent (No. MUST-19010408, purity over 98%) was obtained from Chengdu Man Site Biotechnology Co., Ltd. (Chengdu, China). The Rutin (RU) standard agent (No. L-001-181216) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Cholesterol (No. B80859) and soybean lecithin (No. SY-S1-190601) purchased from A.V.T (shanghai, China) Pharmaceutical Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) L2880 was obtained from Sigma-Aldrich Co., Ltd. (Missouri, USA). BCA protein analysis kit (No. P0012S) was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Mouse TNF-α ELISA kit (96T, NO. 21I333) and Mouse IL-1β ELISA kit (96T, NO21T361) were purchased from Excell Bio Co., Ltd. (Shanghai, China). Primary antibodies against TLR4, p-JNK, and p-ERK were purchased from Affinity Biosciences, OH, USA. p-p65 (phospho S536), p65, ERK, and β-actin were all from Abcam (Cambridge, Britain). JNK was obtained from Proteintech Group, Inc. (Chicago, USA). HRP-Goat anti Rabbit and HRP-Goat anti mouse were both from Abcam (Cambridge, Britain). Water was ultrapure water, methanol and acetonitrile were chromatographic grade, and other reagents were analytical grade.
8
Western Blot Analysis of EMT Markers
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The detailed experimental procedures were as described previously 20 (link). Briefly, RIPA lysis buffer was used to extract proteins from cell samples and tissues. Then we separated equal amounts of lysate protein by SDS-PAGE, and then transferred them onto a PVDF membrane. The following primary antibodies were used: HRC (Sigma-Aldrich, SAB1303636), c-Raf (Affinity Biosciences, Cincinnati, OH, USA; AF6065), phosphorylated (p)-c-Raf (Affinity Biosciences, AF3065), MEK (Affinity Biosciences, AF6385), p-MEK (Affinity Biosciences, AF8035), ERK (Affinity Biosciences, AF0155), p-ERK (Affinity Biosciences, AF1015), N-cadherin (Cell Signaling Technology, Danvers, MA, USA; #13116S), Calmodulin (Cell Signaling Technology, #S35944), Vimentin (Cell Signaling Technology, #5741S), E-cadherin (Cell Signaling Technology, #3195S), Snail (Cell Signaling Technology, #S3879), Slug (Cell Signaling Technology, #9585S), and GAPDH (Abcam, Cambridge, MA, USA; ab9485).
9
Immunohistochemical and Immunofluorescent Staining
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For immunohistochemically staining, sections were incubated by primary antibody RUNX2 (Abcam, ab76956) overnight at 4°C after standard protocol. Horseradish peroxidase-streptavidin detection system was used to detect immunoreactivity and counterstaining with hematoxylin. Immunofluorescent staining was performed using a standard protocol, we incubated sections with primary antibodies: SDF-1 antibody (Santa Cruz, sc-74271), osterix (Santa Cruz, sc-393060), p-ERK (Affinity Bioscience, AF1015), CD31 (Abcam, ab222783), and Endomucin (Emcn, Santa Cruz, sc-65495) overnight at 4°C. A second antibody conjugated with fluorescence was added to the sample sections and incubated for 1 h at room temperature (RT).
10
Western Blot Analysis of EMT Markers
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RIPA buffer was used to extract total protein from cells for 30 min, after which protein was separated via SDS-PAGE and transferred onto an Immobilon-PVDF membrane (Millipore Corporation, MA, USA). Blots were blocked for 2 h with 5% non-fat milk prior to overnight incubation with appropriate primary antibodies at 4°C overnight. Blots were then rinsed with TBS-T and probed with secondary peroxidase-conjugated antibodies prior to protein band visualization with a chemiluminescence reagent (Millipore Corporation). Antibodies used for this study were specific for E-cadherin (#3195, CST), N-cadherin # (22018-1-AP, Proteintech), FLOT-2 (#28208-1-AP, Proteintech), Vimentin (#5741, CST), MEK (#11049-1-AP, Proteintech), p-MEK (#28930-1-AP, Proteintech), p-ERK (#AF1015, Affinity), ERK (#67170-1-Ig, Proteintech), CCND1 (#BF0127, Affinity) CDK4 (#DF6102, Affinity), CDK6 (#DF6448, Affinity).
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