Under Isoflurane anesthesia (3% induction, 1.5–2% for surgery and 1.5% maintenance in a 65:35 mixture of N2O:O2), animals were infused each chemical into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; -3.5 mm depth to the bregma) with a brain infusion kit 1 and an Alzet 1003D osmotic pump (Alzet, United States) for 3 days. Osmotic pump contained (1) control IgG (Abcam, #ab37425, United Kingdom, 50 ug/ml) + vehicle, (2) control IgG + SB202190 (a p38 MAPK inhibitor, 0.3 mg/ml), (3) anti-67-kDa LR IgG (Abcam, #133645, United Kingdom, 50 ug/ml) + vehicle and (4) anti-67-kDa LR IgG (Abcam, #133645, United Kingdom, 50 ug/ml) + SB202190 (0.3 mg/ml). In pilot study and our previous studies (Kim et al., 2016 (link); Ko and Kang, 2017 (link)), each compound treatment did not show behavioral and neurological defects and could not change the seizure susceptibility and seizure severity in response to PILO in normal animals. Three days after surgery (infusion), animals were used for Western blot and immunohistochemistry.
Control igg
Manufactured by Abcam
Sourced in United Kingdom, United States
Control IgG is a non-specific immunoglobulin G (IgG) used as a control in various immunoassays and research applications. It serves as a reference to ensure the specificity of target antibody binding and to help determine background signal levels.
Automatically generated - may contain errors
Lab products found in correlation
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (6 mentions)
Protein a sepharose beads, by Abcam (3 mentions)
Donkey anti mouse igg hrp, by Thermo Fisher Scientific (2 mentions)
Protein g magnetic dynabeads, by Thermo Fisher Scientific (2 mentions)
Ez magna rip kit, by Merck Group (2 mentions)
Mtco1, by Abcam (2 mentions)
Pdgf aa blocking antibody, by Merck Group (2 mentions)
Anti 67 kda lr igg, by Abcam (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
M mlv rt kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Control mirna, by Thermo Fisher Scientific (1 mentions)
Taqman assay kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti mouse and anti rabbit igg conjugated horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Anti 67lr igg, by Abcam (1 mentions)
Cebpd, by Santa Cruz Biotechnology (1 mentions)
Cebpa, by Santa Cruz Biotechnology (1 mentions)
50 protocols using control igg
1
Intracerebroventricular Infusion of Compounds
2
Signaling Pathway Modulation Assay
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Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase as well as rabbit monoclonal antibodies (mAbs) specific for WISP-3, p38, p-p38 (Tyr182), c-Src, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody specific for p-Src (Tyr527) was purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). A VEGF-A ELISA kit was obtained from PerpoTech (Rocky Hill, NJ, USA). Control IgG and rabbit mAb specific for VEGF-A were obtained from Abcam (Cambridge, MA, USA). Control miRNA, miR-452 mimic, MMLV RT kit, Trizol and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The TaqMan MicroRNA Reverse Transcription kit and the TaqMan assay kit were obtained from Thermo Fisher Scientific (Grand Island, NY, USA). c-Src, p38 and control siRNA were purchased from Dharmacon Research (Lafayette, CO, USA). PP2 and SB203580 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
3
Intracerebral Infusion of Inhibitors via Osmotic Pumps
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Under Isoflurane anesthesia (3% induction, 1.5–2% for surgery and 1.5% maintenance in a 65:35 mixture of N2O:O2), animals were infused each chemical into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth to the bregma) with a brain infusion kit 1 and an Alzet 1003D or Alzet 1007D osmotic pump (Alzet, Cupertino, CA, USA) for 3- and 7 days, respectively.
Alzet 1003 osmotic pump contained (1) control IgG (Abcam, #ab37415, UK, 50 ug/mL) + vehicle, (2) anti-67LR IgG (Abcam, #133645, UK, 50 ug/mL) + vehicle, (3) anti-67LR IgG + SB202190 (a p38 MAPK inhibitor, 0.3 mg/mL), (4) anti-67LR IgG + wortmannin (a PI3K inhibitor, 0.1 nmol), (5) anti-67LR IgG + 3-chloroacetyl indole (3CAI, an AKT inhibitor, 25 μM), and (6) 67LR IgG + U0126 (an ERK1/2 inhibitor, 25 μM). Three days after surgery (infusion), animals were used for Western blot and immunohistochemistry.
Alzet 1007D osmotic pump contained (1) vehicle, (2) SB202190 (0.3 mg/mL), (3) wortmannin (0.1 nmol), (4) 3CAI (25 μM), and (5) U0126 (25 μM). In our previous studies [14 (link),16 (link),17 (link),18 (link),30 (link),31 (link)], each treatment did not show behavioral and neurological defects and could not change the seizure susceptibility and seizure severity in response to pilocarpine. Three days after surgery, this group was induced with SE by lithium chloride (LiCl)-pilocarpine.
Alzet 1003 osmotic pump contained (1) control IgG (Abcam, #ab37415, UK, 50 ug/mL) + vehicle, (2) anti-67LR IgG (Abcam, #133645, UK, 50 ug/mL) + vehicle, (3) anti-67LR IgG + SB202190 (a p38 MAPK inhibitor, 0.3 mg/mL), (4) anti-67LR IgG + wortmannin (a PI3K inhibitor, 0.1 nmol), (5) anti-67LR IgG + 3-chloroacetyl indole (3CAI, an AKT inhibitor, 25 μM), and (6) 67LR IgG + U0126 (an ERK1/2 inhibitor, 25 μM). Three days after surgery (infusion), animals were used for Western blot and immunohistochemistry.
Alzet 1007D osmotic pump contained (1) vehicle, (2) SB202190 (0.3 mg/mL), (3) wortmannin (0.1 nmol), (4) 3CAI (25 μM), and (5) U0126 (25 μM). In our previous studies [14 (link),16 (link),17 (link),18 (link),30 (link),31 (link)], each treatment did not show behavioral and neurological defects and could not change the seizure susceptibility and seizure severity in response to pilocarpine. Three days after surgery, this group was induced with SE by lithium chloride (LiCl)-pilocarpine.
4
Chromatin Immunoprecipitation (ChIP) Protocol
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DNA and associated proteins on chromatin in cultured cells were crosslinked by 1% formaldehyde for 15 min at 37 °C. Cells were then scraped and collected in cellular lysis buffer (5 mM Pipes, 85 mM KCl, 0.5% NP-40, and protease inhibitors). Cytoplasmic lysates were discarded and nuclear components were resuspended in nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.2% SDS, and protease inhibitors), and sonicated for 10 min (Covaris). Approximately 4 mg of CEBPA (Cat# sc-365318, Santa cruz), CEBPD (Cat# sc-365546, Santa Cruz Biotechnology) or control IgG (Cat# ab6715, Abcam) were incubated with 25 mL of protein G magnetic beads for 6 h at 4 °C, and then incubated with 100 mg of cleared chromatin overnight at 4 °C. After three washes, immunoprecipitated material was eluted at 55 °C for 1 h with 10 µg/mL proteinase K, and then decrosslinked at 65 °C for 4 h. The primer sequences used for ChIP-qPCR were listed in Supplementary Table S4 .
5
RIP Assay for AGO2 Binding Analysis
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The RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). The lysate from 3 × 107 cells was prepared with 100 μl of RIP lysis buffer. After centrifugation, the supernatant was collected and incubated with RIP wash buffer, and the subsequent procedures were performed according to the manufacturer's instructions. The following antibodies were used for the RIP assay: anti-AGO2 (1 : 20) and control IgG (1 : 20) (Abcam). qPCR was used to detect enriched RNA.
6
ChIP Assay for Transcription Factor Binding
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For Chromatin immunoprecipitation assay (ChIP) cells were cross-linked with a final concentration of 1% formaldehyde for 10 min at 37 °C, then washed and harvested in SDS lysis buffer (10% SDS; 0.5 M EDTA; 1 M Tris-HCl; containing proteinase inhibitor cocktail from Sigma-Aldrich, St. Louis, MO, USA) and sheared by sonication to fragment DNA. Samples were immunoprecipitated with 10 µg of anti-KLF6 antibody (polyclonal antibody KLF6 (R-173) or monoclonal antibody KLF6 (E-10) (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-histone H3 antibody (Abcam, Cambridge, UK) or control IgG (Abcam) and protein-A/G agarose beads (Santa Cruz Biotechnologies). Following removal of cross-linked DNA/protein complexes by Proteinase K (Qiagen, Hilden, Germany) treatment, immunoprecipitated DNA was purified using QIAamp DNA Mini Kit (Qiagen) and used for PCR with ATG7 or BECLIN1 primers (Supplementary Table S3 ), encompassing the promoter region −200 bp to −400 bp upstream of transcriptional start site to amplify immunoprecipitated DNA, PCR products were visualized on an agarose gel.
7
ChIP-qPCR Assay for NRF2 and BMAL1
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Following homogenization, the kidney tissue was cross-linked with 1% (v/v) formaldehyde for 10 min at 37˚C and cell nuclei lyzed using Bioruptor ultrasound (500 bp; 25% power; 4.5 sec impact, 9 sec interval, 14 times in total) to obtain chromatin. The chromatin was incubated with the following antibodies at 4˚C overnight as follows: 1 µg/ml anti-NRF2 (cat. no. ab137550; Abcam), 4 µg/ml anti-BMAL1 (cat. no. ab231793; Abcam), and 1 µg/ml control IgG (cat. no. ab172730; Abcam). Target DNA fragment were obtained by protein G magnetic Dynabead (cat. no. 10003D; Life Technologies) immunoprecipitation, then reverse chromatin cross-linking with 5 M NaCl overnight at 65˚C and digestion by protease K (cat. no. 1.24568; Sigma-Aldrich; Merck KGaA), after which the DNA was eluted. Using RT-qPCR technology, the E-BOX (forward 5'-GAGCCCAGGGCACGTGGGAGAAGTGG-3' and reverse, 5'-CCACTTCTCCCACGTGCCCTGGGCTC-3') of the promoter region was amplified and quantified.
8
Immunoprecipitation of Cytoskeleton Proteins
9
Regulation of Furin Expression by CEBPβ
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SH-SY5Y cells were treated with 0.2 μM dPPA for 72 h, and then, ChIP analysis was performed using ChIP assay kits (Beyotime) according to the manufacturer's recommendations with a CEBPβ antibody (Santa Cruz Biotechnology, sc-9314) and control IgG (Abcam). The input control DNA or immunoprecipitated DNA was then subjected to PCR amplification using primers specific to the furin promoter (forward primer 5′-ACCAGAGCCCAGCGTTCAGCAG-3′ and reverse primer 5′-CATGGCCAGCCAGTGCTGCCA-3′). The PCR products were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide staining.
10
Immunoprecipitation of PARK2 and BECN1
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Fibroblasts were washed twice, harvested in PBS, and then lyzed in ice-cold IP lysis buffer containing 50 mM Tris, 1 mM EDTA, 0.5% NP-40, 150 mM NaCl, 10% glycerol and 1 mM PMSF for 30 min. Total cell extracts were centrifuged for 30 min at 12,000 rpm at 4 °C, then the protein A/G agarose beads (Beyotime Bio, Shanghai, China) or control IgG (Santa Cruz) was incubated with the supernatant for 1 h as a pretreatment. Lysates were incubated overnight with anti-PARK2 monoclonal antibody (Abcam, No. ab179812), anti-BECN1 monoclonal antibody (Abcam, No. ab207612) or control IgG and protein A/G agarose beads on rotary shaker at 4 °C. The immunoprecipitated complexes were collected by centrifugation at 3000 rpm for 5 min at 4 °C, and the beads were washed three times with IP lysis buffer and resuspended in 2 × SDS loading buffer. Subsequently, the immunoprecipitates complexes were eluted from the beads at 99 °C for 5 min. The eluted proteins were determined by Western blot analysis with indicated antibodies.
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