Total RNA was isolated using the Quick-RNA MiniPrep kit (Zymo Research). Briefly, Arabidopsis seedlings (~60 mg) were ground in extraction buffer and processed following the manufacturer’s protocol. Reverse transcription was performed using M-MLV RTase (Promega) using anchored oligo dT. For qPCR, the FastStart Essential DNA Green Master mix was used with a LightCycler 96 (Roche Applied Science). Relative transcript levels were determined by normalizing with PP2A (At1g13320). Immunoblot analyses were performed using rabbit anti-RGA antiserum (DU176, 1:10,000 dilution)11 (link), horseradish peroxidase (HRP)-conjugated anti-FLAG M2 mouse monoclonal (Sigma Aldrich A8592, 1:10,000 dilution) and mouse HRP-anti-MYC monoclonal antibodies (BioLegend #626803, 1:1,000 dilution), mouse anti-HA monoclonal antibody (BioLegend 901503, 1:1,000 dilution), rabbit anti-H2A monoclonal antibody (Abcam #ab177308, 1:1,000 dilution) and rabbit anti-Histone 3 polyclonal antibody (Abcam #ab1791). HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch #715-035-150) was used for anti-HA at 1:10,000 dilution. HRP-conjugated goat anti-rabbit IgG (Thermo-Fisher #31462) was used to detect anti-RGA and anti-HA at 1:10,000 dilution. Y2H assays were performed as described previously45 (link).
Ab177308
Manufactured by Abcam
Sourced in United States
Ab177308 is a primary antibody that can be used for immunohistochemistry applications. The product is manufactured by Abcam.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (2 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Hrp conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
M mlv rtase, by Promega (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Ab52599, by Abcam (1 mentions)
Ab10158, by Abcam (1 mentions)
Ab5103, by Abcam (1 mentions)
Ht501, by Transgene (1 mentions)
Af3667, by R&D Systems (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab125066, by Abcam (1 mentions)
Ab13248, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
4 protocols using ab177308
1
Molecular Techniques for Arabidopsis Transcriptomics
2
Neutrophil and Lung Tissue Protein Analysis
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After collection of human neutrophils and murine lung tissues (6 h and 24 h post-challenge), the samples were homogenized in RIPA lysis buffer containing protease inhibitor cocktails on ice. An equal amount of protein per sample was subjected to 12% SDS-PAGE and the proteins were blotted onto a Hybond-enhanced chemilumescent nitrocellulose membrane, which were then incubated with the primary antibodies (anti-human/mouse myeloperoxidase/MPO (AF3667, R&D), anti-histone H3 (citrulline R2 + R8 + R17) (ab5103, abcam), anti-histone H2A (ab177308, abcam), anti-histone H2B (ab52599, abcam), anti-histone H3 (ab1791, abcam), anti-histone H4 (ab10158, abcam), anti-His (HT501, Transgen Biotech), Ra-anti-AprA, Hu-anti-AprA), then subsequently with appropriate HRP-conjugated secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG; rabbit anti-goat IgG,; goat anti-human IgG, Jackson ImmunoResearch Laboratories). Equal loading was confirmed by probing for β-actin (HC201, Transgen Biotech).
3
Western Blot Analysis of Antioxidant Proteins
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We performed western blot as previously recorded [27 (link)]. Proteins were extracted from lung tissues or cells and were then lysed in a RIPA buffer containing protease and phosphatase inhibitors. The BCA kit quantified the protein content (Beyotime, Shanghai, China). 50 μg of protein samples from each group (Nrf2 in the nuclear fraction, HO-1 in the cytosolic fraction, and others from both fractions) was electrophoresed in a 10% SDS-PAGE gel and transferred to a PVDF membrane. The blots were incubated with the primary antibodies against GPX4 (1: 1000, ab125066, Abcam), ACSL4 (1: 1000, ab155282, Abcam), Nrf2 (1: 1000, ab62352, Abcam), HO-1 (1: 1000, ab13248, Abcam), histone H2A (1: 1000, ab177308, Abcam), or GAPDH (1: 1000, ab181602, Abcam) overnight at 4°C after blocking with 5% nonfat dry milk. After that, the blots were washed in TBST and incubated for 2 hours at room temperature with a secondary antibody. The bands were identified by an ECL system and an ECL kit (Beyotime, Shanghai, China), and band intensities were checked using the ImageJ software.
4
Immunofluorescence Analysis of DNA Damage
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Cells were seeded on cell climbing pieces, followed by fixed by 4% paraformaldehyde, and washed by Phosphate‐Buffered Saline (PBS) solution for three times. Then, cells were incubated with 0.5% Triton X‐100 for 15 min, and blocked in FBS for 60 min. Primary antibodies (γH2AX, purchased from Abcam, Cambridge, MA, USA, ab81299; SSRP1, purchased from Abcam, ab26212; H2A, purchased from Abcam, ab177308; H2B, purchased from Abcam, ab1790) were used and incubated at 4°C overnight. Secondary antibodies (ThermoFisher, MA, USA) were used and incubated at room temperature for 1 h. 4′,6‐diamidino‐2‐phenylindole (DAPI) was used for cell nucleus staining. Pictures were taken by laser confocal microscopy.
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