Proteins in total cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). PVDF was incubated with primary antibodies against eIF5a (ab32443; Abcam), CBL (ab52855; Abcam), or β‐actin (A5441; Sigma‐Aldrich LLC) overnight at 4°C. Subsequently, horseradish peroxidase‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG was applied at room temperature for 2 hours. Membranes were analyzed by a ChemiDoc XRS + image analyzer (Bio‐Rad).
Ab32443
Manufactured by Abcam
Sourced in United States
Ab32443 is a laboratory equipment product manufactured by Abcam. It is designed for general laboratory use, but its core function is not available without further details.
Automatically generated - may contain errors
Lab products found in correlation
Ab108928, by Abcam (2 mentions)
Ab109376, by Abcam (2 mentions)
Ab216876, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Chemidoc xrs image analyzer, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab53154, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab6728, by Abcam (1 mentions)
Cisplatin, by Enzo Life Sciences (1 mentions)
Anti kras antibody, by Santa Cruz Biotechnology (1 mentions)
Pa2081a, by Tissue Array (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
8 protocols using ab32443
1
Immunoblotting Analysis of Cellular Proteins
2
Protein Expression Analysis in Cell Lysates
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Tissue homogenates and cells were treated with RIPA lysates (Beyotime, China), and supernatants containing proteins were collected by centrifugation. Protein content was assessed by BCA detection kit (EMD Millipore). SDS-PAGE was performed with the same amount of protein samples in each lane, and then the isolated proteins were electrotransferred to PVDF membrane (Millipore). 5% skimmed milk powder was used for blocking the membranes, and then the corresponding primary antibody (eIF5A (1 : 1000, ab32443, Abcam); FANCD2 (1 : 1000, ab108928, Abcam); SLC7A11 (1 : 1000, ab216876, Abcam); HSPB1 (1 : 1000, ab109376, Abcam); Bax (1 : 1000, ab53154, Abcam); Bcl-2 (1 : 1000, ab32124, Abcam); cleaved caspase-3 (1 : 1000, ab32042, Abcam); cytochrome C (1 : 1000, ab133504, Abcam); β-actin (1 : 1000, ab8226, Abcam)) was applied overnight at 4°C. Following that, the membranes were treated for 2 h with the corresponding secondary antibody (Goat Anti-Rabbit IgG H&L (1 : 2000, ab6721, Abcam) and Rabbit Anti-Mouse IgG H&L (1 : 2000, ab6728, Abcam)). The transfer protein on membranes was developed with electrochemiluminescence (ECL, Thermo Fisher Scientific, USA)). Grayscale of the strips was assessed by ImageJ 1.48v software (NIH).
3
Antibody Characterization for Protein Expression
4
Immunohistochemical Analysis of Cellular Markers
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Slices were dewaxed and hydrated. Endogenous catalase was removed by H2O2. The slices were blocked for 30 min at 37°C with 5% BSA solution (P0220, Beyotime). Following that, the sections were reacted with primary antibody [eIF5A (1 : 250, ab32443, Abcam); FANCD2 (1 : 100, ab108928, Abcam); SLC7A11 (1 : 500, ab216876, Abcam); HSPB1 (1 : 500, ab109376, Abcam)] overnight at 4°C. The sections were incubated with the secondary antibody for 30 min. Then the slides were treated for 5 min with diaminobenzidine (DAB, Beyotime, China). After restaining for 5 min with hematoxylin, the slices were dehydrated, made transparent, and finally sealed with neutral resin. The results were observed with an optical microscope (Olympus Corporation).
5
Culturing Pancreatic Cancer Cell Lines
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The human PC cell lines Panc‐1 and BxPc‐3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotic/antimycotic (Life Technologies, Carlsbad, CA, USA). The cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Antibodies against sHH (ab53281), SMO (ab5694), PTCH (ab53715), Gli‐1 (ab49314), GAPDH (ab8245) and EIF5A (ab32443) were purchased from Abcam (Cambridge, MA, USA). Recombinant sHH was obtained from R&D Systems (Minneapolis, MN, USA).
6
Immunohistochemical Analysis of eIF5A in Pancreatic Cancer
7
Western Blot Profiling of Apoptosis and EMT
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Total proteins were extracted using RIPA buffer, and quantified with a BCA Protein Assay Kit (Tiangen, Beijing, China). 20 μg of proteins were taken for SDS-PAGE electrophoresis, and then electrotransferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h, incubated with the specific primary antibody at 4 °C overnight and incubated with the second antibody for 1 h at room temperature. ECL development was performed after washing the membrane through TBST.
The primary antibodies used in this study were as follow: anti-Bcl2 (1:1000, ab32124), anti-Bax (1:1000, ab32503), anti-p53 (1:1000, ab32389), anti-E-cadherin (1:1000, ab194982), anti-SNAL2 (1:1000, ab180714) and anti-eIF5A (1:1000, ab32443) were rabbit monoclonal antibodies and purchased from Abcam (Cambridge, MA, USA). Anti-Caspase3 (1:1000, ab13847) and anti-N-cadherin (1:1000, ab18203) were rabbit polyclonal antibody and purchased from Abcam. Anti-GAPDH (1:500, ab8245, Abcam) and anti-Vimentin (1:1000, ab8978) were mouse monoclonal antibodies and purchased from Abcam.
The primary antibodies used in this study were as follow: anti-Bcl2 (1:1000, ab32124), anti-Bax (1:1000, ab32503), anti-p53 (1:1000, ab32389), anti-E-cadherin (1:1000, ab194982), anti-SNAL2 (1:1000, ab180714) and anti-eIF5A (1:1000, ab32443) were rabbit monoclonal antibodies and purchased from Abcam (Cambridge, MA, USA). Anti-Caspase3 (1:1000, ab13847) and anti-N-cadherin (1:1000, ab18203) were rabbit polyclonal antibody and purchased from Abcam. Anti-GAPDH (1:500, ab8245, Abcam) and anti-Vimentin (1:1000, ab8978) were mouse monoclonal antibodies and purchased from Abcam.
8
Protein Expression Analysis of Inflammasome Pathway
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Lysed samples collected by Radio-Immunoprecipitation Assay buffer (Beyotime) were measured by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane which was then covered with 5% skim milk for overnight incubation with anti-pro-caspase-1 (ab179515, Abcam), gasdermin D (GSDMD; 20770-1-AP, Proteintech), plasma-membrane-bound pannexin-1 (PANX1; 12595-1-AP, Proteintech), p-p65 (3033, Cell Signaling Technology), p65 (ab86299, Abcam), eIF5A (ab32443, Abcam), and glyceraldehyde-3phosphate dehydrogenase (ABclonal, A19056, 1:1000). Next, horseradish peroxidase-conjugated secondary antibody was added for 1.5 h and enhanced chemiluminescence-developed bands were imaged using Tanon 5200 (Tanon, China) and analyzed using ImageJ software.
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