ChIP assay was performed with a ChIP assay kit (Millipore) according to the manufacturer's instructions. Chromatin was extracted from ARP-1 cells. Anti-NF-κB p65 antibody and isotype control (Cell Signaling Technology) were used for the chromatin immunoprecipitation. The precipitated DNA was analyzed by qPCR with the following primer sets for the region surrounding the NF-κB binding sites at the beclin 1 promoter: F 5′-AGA CCA GCC TGG CCA AAA TGG T-3′ and R 5′-TGA GAT GGA GTT TCC TTC TGT CG-3′. Values were subtracted from control IgG values and normalized to corresponding input control.
Isotype control
Manufactured by Cell Signaling Technology
Sourced in United States
Isotype control is a type of laboratory reagent used in flow cytometry and other immunoassays to determine the specificity of an antibody. It is a control antibody that shares the same isotype, but does not recognize the target antigen. Isotype controls are used to establish the level of non-specific binding of the primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
Chip assay kit, by Merck Group (1 mentions)
Phosflow perm buffer 3, by BD (1 mentions)
Phosflow lyse fix buffer, by BD (1 mentions)
Biotin anti cd28, by BD (1 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
Ez magna chip g kit, by Merck Group (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Anti gli1, by Novus Biologicals (1 mentions)
Cellmask plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Macsplex exosome kit, by Miltenyi Biotec (1 mentions)
Alexa fluor 488 conjugated ki 67 rabbit mab, by Cell Signaling Technology (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Bioruptor ucd 200, by Diagenode (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Magnetic chip kit, by Thermo Fisher Scientific (1 mentions)
Phospho p44 42 mapk erk1 2 rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Lsri flow cytometer, by BD (1 mentions)
Zombie live dead stain, by BioLegend (1 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
10 protocols using isotype control
1
Chromatin Immunoprecipitation of NF-κB
2
Phosphorylation of AKT in Thymocytes
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Thymocytes were rested at 37°C for 20 min to allow for dephosphorylation of AKT. Cells were then stained with LIVE/DEAD Fixable Aqua (Thermo Fisher) at 4°C for 20 min. Cells were washed and incubated with 5 µg/ml anti-CD3-biotin (Invitrogen) and 5 µg/ml anti-CD28-biotin (BD Biosciences) along with extracellular markers on ice for 15 min. To stimulate the cells, the cells were incubated with 20 µg/ml of streptavidin (Thermo Fisher) for the indicated time points (0, 2, 5, or 10 min) at 37°C before being fixed with Phosflow Lyse/Fix Buffer (BD Biosciences) for 10 min at 37°C. Cells were washed and resuspended with prechilled Phosflow Perm Buffer III (BD Biosciences) dropwise and incubated on ice for 1 h. Cells were washed twice before being stained with either isotype control (Cell Signaling Technology) or pAKT (pS473) (Cell Signaling Technology) overnight at 4°C.
3
Profiling KRAS Promoter Regulation
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Chromatin immunoprecipitation assays were carried out using the EZ-Magna ChIPTM G Kit (Millipore 17-611) according to the manufacturer’s instruction. H358 cells were treated with a combination of 10 μM ARS-1620 and 10 μM sonidegib for 48 h. Nuclei were isolated from the cells and sonicated to shear the DNA, which was distributed around 0.2 kb to 1 kb on 1% agarose gel. Chromatin was immunoprecipitated with anti-GLI-1 (Novus, NB 600-600), anti-histone H3 (Cell Signaling, 4620) or an isotype control (Cell Signaling, 2729). The complexes were collected on Protein G Magnetic beads and subsequently extracted from the beads. Bound DNA was purified and amplified by qRT-PCR with primers that amplify 204-bp (−1262 to −1059), 618-bp (-1078 to -460), 260-bp (-594 to -335), 354-bp (-354 to -1), and 117-bp (−1205 to −1089) regions (Table 1 and Fig. S4 ) of the KRAS promoter.
4
Extracellular Vesicle Protein Analysis
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EV proteins were extracted and analyzed by western blotting as previously reported (Choi et al., 2020 (link)) with few modifications as detailed in Supplementary Materials. EV surface marker analysis was performed by MACSPlex Exosome kit (Miltenyi Biotech; no. 130‐108‐813) following the manufacturer's protocol tube for overnight capture on tubes. HER2 protein detection on EV was performed by staining samples with HER2/ErbB2 (29D8)—PE‐conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies). HER2‐positive EV were identified on an Amnis ImageStream X MkII (Luminex).
5
Confocal Microscopy Analysis of Cell Proliferation
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Confocal microscopy analysis was prepared in the following manner. One round platinum lined cover glass 12 mm in diameter was placed in each well of a 12-well plate then coated with fibronectin and collagen II for at least 12 h. Cells were then seeded on cover glass inside of wells to normalize treatment to MTT assays and IncuCyte analysis. After selected drug treatment, CAP treatment, or combination treatment cultures were fixed with ice cold (− 20 °C) methanol for 10 min. Then cells were stained with Alexa Fluor 488-conjugated Ki-67 Rabbit mAb (Cell Signaling Technology, #11882) or Isotype control (Cell Signaling Technology, #4340) antibodies according to Immunofluorescence General Protocol by Cell Signaling Technology (Danvers, MA, USA). Cells were incubated overnight at 4 °C protected from light. The cover slides were then carefully moved onto glass slides and covered with Antifade Mounting Reagent with DAPI (Vector Laboratories, H-1500) drops and then a 1 mm cover slide. Slides were allowed to cure for up to 2 nights in a 4 °C refrigerator then sealed with clear nail polish. Images were taken with Zeiss Confocal 510 LSM (Oberkochen, Germany), analyzed with Zeiss ZenLite (2012) software, and Ki-67 positivity was calculated in Microsoft Excel 2019 (Redmond, WA, USA).
6
Chromatin Immunoprecipitation (ChIP) Protocol
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Cultured cells were washed with PBS and cross-linked with 1% formaldehyde, followed by cell lysis using SDS buffer. Lysate was sonicated with Bioruptor UCD-200 (Diagenode), followed by incubation with Dynabeads (Invitrogen) conjugated with rabbit-derived antibodies: SPI1, STAT1, or isotype control (all Cell Signaling Technology). Protein/DNA/bead complexes were washed with radioimmunoprecipitation assay buffer ( RIPA buffer), RIPA + NaCl, LiCl, and TE buffer. Protein/DNA complexes were eluted with elution buffer. Reverse cross-linking was performed overnight at 65°C, followed by DNA purification and qPCR. ChIP signal was normalized to total chromatin input (percent input), which was calculated as 100 × 2(CTinput-CTtarget).
Primers (Integrated DNA Technologies) were as follows: Promoter, (f) 5′-FW CAG GAA ACG CCT AAG GAC TC-3′ and (r) 5′-AAGGGATGGGAGGTGAATCG-3′; Enhancer, (f) 5′-ATCTCTCTCACCTTTGGCGC-3′ and (r) 5′-ATGCAGTGAGTAGAGGCTGG-3′; Intronic, (f) 5′-TCTTGTCTCACGGCACCTTG-3′ and (r) 5′-AAACAAGACACAGCTCCCGC-3′; Negative Control, (f) 5′-GGCTACCCAGAGTAATGACC-3′ and (r) 5′-GAAGAAAGGGACATGGCAGC-3′.
Primers (Integrated DNA Technologies) were as follows: Promoter, (f) 5′-FW CAG GAA ACG CCT AAG GAC TC-3′ and (r) 5′-AAGGGATGGGAGGTGAATCG-3′; Enhancer, (f) 5′-ATCTCTCTCACCTTTGGCGC-3′ and (r) 5′-ATGCAGTGAGTAGAGGCTGG-3′; Intronic, (f) 5′-TCTTGTCTCACGGCACCTTG-3′ and (r) 5′-AAACAAGACACAGCTCCCGC-3′; Negative Control, (f) 5′-GGCTACCCAGAGTAATGACC-3′ and (r) 5′-GAAGAAAGGGACATGGCAGC-3′.
7
Chromatin Immunoprecipitation Analysis of CD8 T Cell Activation
8
Phospho-ERK Expression in Sebaceous Gland Hyperplasia
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Five SGH with a proven activating mutation in HRAS, KRAS or EGFR, 5 SGH and 5 normal sebaceous glands showing an HRAS, KRAS and EGFR wildtype status in the genetic analyses performed were evaluated for phosphorylated ERK using immunohistochemistry. Phospho-p44/42 MAPK (Erk1/2) Rabbit monoclonal antibody detecting phosphorylation at Threonin 202/ Tyrosine 204 (Cell Signaling Technology, Danvers, MA, United States, #4370, dilution 1:50) was applied according to the manufacturer's instructions. Isotype control was performed to rule out unspecific staining (Cell Signaling Technology, Danvers, MA, United States, #3900). The overall Phospho-p44/42 MAPK staining intensity (not the frequency of positive tumour cells) was scored 0 (negative), 1+ (weak), 2+ (strong), and 3+ (very strong) by 2 individual investigators.
9
Macrophage Flow Cytometry Analysis
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Macrophages were lifted by gentle cell scraping (catalog no. 83.1830; Sarstedt) into PBS, washed once with PBS, and incubated with Zombie live/dead stain (BioLegend) at 4 °C. After washing, cells were fixed in 4% formaldehyde, washed, and permeabilized in 90% ice-cold methanol. Samples were washed and blocked with Human Fc Block for 10 min at 4 °C followed by 30-min incubation at 4 °C with directly conjugated 4G2-AF647, AF488-Phospho-Stat1 (Tyr701) (58D6), PE-Stat1 (D1K9Y), or isotype controls (Cell Signaling). Samples were centrifuged, washed twice with PBS + 0.5% BSA, and analyzed by flow cytometry on a BD LSRI flow cytometer.
10
Quantification of Epidermal Cleaved Caspase-3
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For detection of cleaved caspase-3 in mouse skin, formalin-fixed, paraffin-embedded sections were heated at 60°C for 1 hour, deparaffinized, rehydrated, and heated at 100°C for 20 minutes in Tris-EDTA buffer (pH 9.0) for antigen retrieval. Slides were washed, treated with 3% hydrogen peroxide in PBS for 5 minutes, blocked in goat serum for 1 hour, and incubated with Human/Mouse Cleaved Caspase-3 (Asp175) antibody (1:100 dilution, MAB835 R&D) overnight at 4°C. Isotype controls (#3900; Cell Signaling Technology) were stained in parallel with each set of slides. All slides were incubated with biotinylated goat anti-rabbit IgG secondary antibody (1:200; Vector Laboratories, Newark, California), followed by incubation with VECTASTAIN Elite ABC reagent (Vector Laboratories) and detection with 3,3’-diaminobenzidine under a light microscope. Slides were counterstained with hematoxylin, dehydrated, and mounted. Images were acquired using a Zeiss microscope at indicated magnifications. Percent epidermal caspase-3+ cells was quantified by manually counting the # of positive cells (brown)/total # of nuclei in the epidermis averaged for three 20x fields of view.
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