Whole lysates from B16-F10 p53+/+ and p53−/− cells were probed for p53 and p21 expression. Cells were treated for 24 hours with 1.5 μmol/L Navtemadlin (to activate p53) or with DMSO control, lysed using 1 × Laemmli lysis buffer, heated at 95°C for 5 minutes, and sonicated (Qsonica Sonicators) for 30 seconds at 20% amplitude. After protein concentrations were determined using the DC Protein Assay (Bio-Rad), lysates were loaded (18 μg/lane) in a 4%–15% polyacrylamide gel (Mini-Protein TGX Stain-Free 12 well, Bio-Rad), and electrophoresed for 45 minutes at 150 V. Proteins were then transferred using a semidry method onto polyvinylidene difluoride membranes for 30 minutes according to manufacturer's instructions (Bio-Rad). Once membranes were blocked for 1 hour with blocking buffer (5% milk made in PBS containing 0.1% Tween-20), they were incubated overnight at 4°C with rabbit anti-mouse p53 (Abcam, EPR20416-124, 1:1,000) or rabbit anti-mouse p21 (Abcam, EPR18021, 1:1,000) primary antibodies made in blocking buffer. Membranes were subsequently washed in PBS containing 0.1% Tween-20, incubated for 1 hour at room temperature with goat anti-rabbit HRP secondary antibody (Dako, 1:1,000), and washed again. Protein expression was detected using chemiluminescence (Clarity Western ECL Substrate, Bio-Rad).
Epr18021
Manufactured by Abcam
Sourced in United Kingdom
EPR18021 is a recombinant monoclonal antibody targeting GOLPH3. The antibody is produced in a mammalian cell line and is suitable for use in various applications, including Western blot, immunohistochemistry, and immunofluorescence, among others.
Automatically generated - may contain errors
Lab products found in correlation
Sonicator, by Qsonica (1 mentions)
Goat anti rabbit hrp secondary antibody, by Agilent Technologies (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Cryostat, by Leica (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Ab188224, by Abcam (1 mentions)
E8f4l, by Cell Signaling Technology (1 mentions)
Epr5368, by Abcam (1 mentions)
3 protocols using epr18021
1
Western Blotting of p53 and p21 in B16-F10 Cells
2
Immunofluorescent Imaging of p21 in Fixed Cryosections
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To prepare sections, mouse organs were fixed in 4% paraformaldehyde at 4°C for 16 hours, followed by cryopreservation pretreatment in 30% sucrose for additional 16 hours. The organs were embedded in OCT (optimal cutting temperature) compound and frozen at −80°C; 16 hours later, blocks were cut into 10-μm sections by using a Leica cryostat. Slide-mounted sections were dried for 16 hours at 25°C. Immunofluorescent detection of p21 was performed by permeabilizing sections with 0.1% Triton X-100 in PBS for 2 min at 25°C, blocking with 10% goat serum for 1 hour at 25°C, and incubating with anti-p21 antibody (Abcam, [EPR18021], ab188224) in 10% goat serum for 16 hours at 4°C. A fluorescent secondary antibody [Invitrogen, goat anti-rabbit Immunoglobulin G (H+L) superclonal, Alexa Fluor 647] was then incubated in 10% goat serum for 1 hour at 25°C, and slides were mounted with ProLong Diamond Antipode Mountant with 4′,6-diamidino-2-phenylindole (Life Technologies) and coverslips. TUNEL assay (In Situ Cell Death Detection Kit, Fluorescein, Roche) was performed following the manufacturer’s instructions. Signals were collected using a fluorescence microscope (BZ-X Analyzer, Keyence) and analyzed with ImageJ.
3
Antibody Immunostaining Protocol
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The primary antibodies used in the present study were: rabbit anti-Cdkn1a (EPR18021 at 1:500 dilution; Abcam, Cambridge, U.K.), rabbit anti-α-SMA (EPR5368, 1 in 8000 dilution; Abcam), rabbit anti-collagen type I (E8F4L, 1 in 400 dilution; Cell Signaling Technology, Danvers, MA, U.S.A.), or goat anti-KIM-1 (AF1817 at 1:500 dilution, R&D systems, Minneapolis, MN, U.S.A.). Irrelevant rabbit and goat primary antibodies were used as controls to ensure the specificity of immunostaining.
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