Co-Immunoprecipitation was performed according to the standardized protocol. The Immunoprecipitation Kit with Protein A+G Magnetic Beads was produced by Beyotime (P2179S, Beyotime, Shanghai, China). Huh-7 cells were treated with cisplatin with a dose of 10 µM for 24 h and washed with PBS three times, and then 100–200 μL inhibitor lysate was added for 10 min on ice. Lysates were centrifuged at 14,000× g at 4 °C for 5 min and incubated with an anti-IRF1 antibody (ab243895, abcam, Burlingame, CA, USA) or anti-CHK1 antibody (25887-1-AP, Proteintech, China) attached to Protein A+G magnetic beads. The protein samples were incubated with protein A+G magnetic beads bound with antibodies overnight at 4 °C on the shaker platform. Next, beads were collected by centrifugation at 3000× g for 5 min at 4 °C, washed in lysis buffer, and then resuspended in SDS gel loading buffer. The protein samples were prepared for western blot analysis.
Ab243895
Manufactured by Abcam
Sourced in United States
Ab243895 is a laboratory equipment product offered by Abcam. It serves as a core function of [PRODUCT_CORE_FUNCTION].
Automatically generated - may contain errors
Lab products found in correlation
Ab52894, by Abcam (1 mentions)
Ab32430, by Abcam (1 mentions)
Ab197772, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Chip it express enzymatic magnetic chromatin immunoprecipitation kit enzymatic shearing kit, by Active Motif (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
A10523, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
5 protocols using ab243895
1
Co-Immunoprecipitation of IRF1 and CHK1
2
Western Blot Analysis of Protein Expression
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Fifty micrograms (50ug) of cell protein were extracted from HBE cells according to previous procedures (23 (link)). In brief, isolated protein from cells or lung tissues were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was incubated bated with primary antibody for 12 hours and next incubated with Horseradish Peroxidase (HRP) conjugated secondary antibody. The following antibodies were used to determine the levels of ITGB4 (ab197772, Abcam, USA), EGFR (ab52894, Abcam, USA), p-EGFR (ab32430, Abcam, USA) and IRF-1 (ab243895, Abcam, USA). GAPDH (ab8245, Abcam, USA) was used as corresponding controls as indicated.
3
ChIP-qPCR Analysis of MICA Promoter
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ChIP was conducted as previously described [14 (link)]. The ChIP-IT® Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit & Enzymatic Shearing Kit purchased from Active Motif (Carlsbad, CA, USA) was used for the ChIP assay. We designed seven pairs of qPCR primers in the human MICA transcript promoter region. Huh-7 cells were harvested after cisplatin was treated with a dose of 10 µM for 6 h. ChIP experimental procedures are performed according to the manufacturer’s instructions. Anti IRF1 antibody (ab243895, abcam, ChIP grade) was used in the experiment. The DNA collected by ChIP was detected by qPCR.
4
Immunostaining for IRF1 and pSTAT1 in MSCs
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MSCs were plated in 6 well plates containing coverslips, cultured for 24 hours, and fixed with 4% PFA in PBS for 20min at 4–8°C. Cells were washed thoroughly with 0.2μm filtered 5% BSA in PBS (blocking solution), and permeabilized by incubation in 0.2% Triton-X-100 in PBS at room temperature for 5min. After washing and incubating with blocking solution for 1 hour, cells were immuno-stained overnight at 4–8°C with rabbit anti-human IRF1 antibody (1:1000, ab243895, Abcam, Cambridge, UK) or rabbit anti-human pSTAT1 (1:400, MA5–15071, Thermo, Waltham, MA) and mouse anti-human STAT1 (1:800, 66545-1, Proteintech, Rosemont, IL) diluted in blocking buffer. Secondary antibody staining was performed using Cy5 Goat anti-rabbit (1:500, A10523, Thermo, Waltham, MA), AF 594 donkey anti-rabbit (1:500, A21207, Invitrogen, Waltham, MA), or AF 647 goat anti-mouse (1:500, A21235, LTC, Carlsbad, CA). Secondary staining solutions also contained Hoechst nuclear stain (1:10,000, 33342, Thermo, Waltham, MA). Cells were visualized with a Nikon Eclipse Ti2 at 60x magnification. Background subtraction was done using ImageJ and was performed identically for each fluorophore.
5
Immunohistochemical Profiling of Liver Tumors
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Each collected tissue specimen was formalin-fixed and paraffin-embedded, and then the tumor and adjacent liver tissue were placed on the same block. Consecutive 5-micron thick sections were cut from each block and mounted on glass slides. After dewaxing, rehydration, and antigen repair, the anti-CHK1 (25887-1-AP), CD8 (66868-1-Ig), and CD56 (14255-1-AP) (Proteintech, Wuhan, China) anti-IRF1 (ab243895, abcam, Burlingame, CA, USA) antibodies were used respectively in IHC staining according to standardized institutional protocols. All slides were counterstained with hematoxylin, dehydrated, and sealed with neutral resin.
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