Cells were harvested and lysed and the supernatants were collected. Tweenty‐five micrograms protein was loaded into each well of 10% or 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Separated proteins were then transferred onto Polyvinylidene fluoride membrane followed by blocking for 1 hour at room temperature in 5% nonfat milk. The blots were then incubated with primary antibody overnight at 4°C (CPNE1; Abcam, Cambridge, MA; ab155675, 1:1000; cleaved caspase‐3; Abcam, ab2302, 1:500; cleaved PARP1; Abcam, ab32064, 1:500; AKT; CST, Danvers, MA, #9272, 1:1000; p‐AKT; CST, #9271, 1:000; β‐actin; CST, #4970, 1:1000). The HRP‐labeled secondary antibody (Beyotime Biotechnology Inc., Shanghai, China; A0208; 1:1000) was incubated with the membrane at 37°C for 1 hour. Finally, the membranes were incubated with an enhanced chemiluminescence detection kit (Millipore, Billerica, MA) for image scanning. β‐actin was used as a loading control.
Ab155675
Manufactured by Abcam
Sourced in United States
Ab155675 is a primary antibody that recognizes the target protein. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab229912, by Abcam (2 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Ab2302, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Diaminobenzidine dab, by Thermo Fisher Scientific (1 mentions)
Ab86305, by Abcam (1 mentions)
Cfx manager machine, by Bio-Rad (1 mentions)
Ab2000, by Abways (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fastquant rt kit, by Tiangen Biotech (1 mentions)
Ab124800, by Abcam (1 mentions)
Ab183094, by Abcam (1 mentions)
Ab31390, by Abcam (1 mentions)
Ab133627, by Abcam (1 mentions)
Ab168372, by Abcam (1 mentions)
Ab169528, by Cell Signaling Technology (1 mentions)
P perk, by Cell Signaling Technology (1 mentions)
P eif2α, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
5 protocols using ab155675
1
Western Blot Analysis of Apoptosis Markers
2
Immunohistochemical Analysis of CPNE1
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Colorectal tumor tissue sections (4 to 7-μm thick) were rehydrated and incubated overnight at 4°C with anti-CPNE1 antibody (ab155675, Abcam, Cambridge, MA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Thermofisher, Bridgewater Township, NJ, USA) at 25°C for 30 mins. Diaminobenzidine (DAB) (Thermofisher, Bridgewater Township, NJ, USA) was used as the HRP substrate.
3
Comprehensive RNA and Protein Analysis
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Total RNA and protein were isolated with TRIzol reagent according to manufacturer's instructions (Invitrogen). Then, polyA+ RNA was reversely transcribed into cDNA with oligo‐(dT) primer (AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN) using FastQuant RT Kit (Tiangen). Gene expression at the RNA level was quantified by qRT‐PCR using 2× SYBR mix (Vazyme). GAPDH served as an internal control. Then, the reaction was run on Bio‐Rad CFX manager machine.
For Western blot, protein was incubated at 95°C for 10 min before performing SDS‐PAGE. The primary antibodies for U2AF1 (ab86305; 1: 2000, Abcam), CPNE1 (ab155675, 1: 1000, Abcam), and GAPDH (AB2000, 1: 5000, Abways) were incubated with membrane for 2 h at room temperature. The blots were then washed and incubated with second antibody (Goat Anti‐Rabbit, M21002L, 1: 5000, Abmart) for 1 h at room temperature. After washing in TBST, blots were exposed by Tanon Chemiluminescent Imaging System.
For Western blot, protein was incubated at 95°C for 10 min before performing SDS‐PAGE. The primary antibodies for U2AF1 (ab86305; 1: 2000, Abcam), CPNE1 (ab155675, 1: 1000, Abcam), and GAPDH (AB2000, 1: 5000, Abways) were incubated with membrane for 2 h at room temperature. The blots were then washed and incubated with second antibody (Goat Anti‐Rabbit, M21002L, 1: 5000, Abmart) for 1 h at room temperature. After washing in TBST, blots were exposed by Tanon Chemiluminescent Imaging System.
4
Protein Expression Analysis in Tissue Lysates
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Tissues or cells were lysed with RIPA buffer. Protein content in lysates was measured using bicinchoninic acid and equal amounts of proteins (20 μg) were separated using SDS-PAGE with β-Actin as a loading control. Separated proteins were transferred to PVDF membranes (Millipore, USA) and subsequently blocked with 5% nonfat dry milk. The membranes were incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used: CPNE1 (1:1000, abcam, ab155675), ATROGIN1 (1:1000, Abcam, ab168372), MuRF1 (1:1000, Abcam, ab183094), MyoG (1:1000, Abcam, ab124800), MyoD (1:1000, Abcam, ab133627), GLB1 (1:1000, Abcam, ab203749), p-eIF2α (1:1000, Cell Signaling Technologies, #9721), eIF2α (1:1000, Abcam, ab169528), p-PERK (1:1000, Cell Signaling Technologies, #3179), PERK (1:1000, Abcam, ab229912), ATF4 (1:1000, Abcam, ab31390) and β-Actin (1:1000, Abcam, ab8226). Protein gray values were measured using Image J software.
5
Verifying CPNE1-PERK Protein Interaction
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To verify the direct binding between CPNE1 and PERK, cells were lysed and incubated with antibodies against CPNE1 (1:1000, abcam, ab155675) at 4 °C overnight. Subsequently, cell lysates were cultivated with protein A/G agarose beads (Santa Cruz, CA, USA) for 2 h. Finally, western blotting with antibodies against PERK (1:1000, Abcam, ab229912) was employed to analyze the respective protein expression in immuno-complexes.
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