Cells were seeded in 12-well plates containing pre-inserted glass slides. Then, the cells were washed with PBS 24 h later at a confluence of 40–50%. Cells were fixed with 4% paraformaldehyde for 30 min afterwards, followed by permeabilization with 0.5% Triton X-100 solution for an additional 20 min. Next, 5% bovine serum albumin was added to function as the blocking buffer. The primary antibodies, anti-CPNE1 (Abcam, UK) and anti-RACK1 (Santa Cruz, USA), and corresponding secondary antibodies conjugated to Cy3 and FITC were used successively.
Anti cpne1
Manufactured by Abcam
Sourced in United Kingdom
Anti-CPNE1 is an antibody product used for research purposes. It is designed to detect the CPNE1 protein, which plays a role in cellular processes. The antibody can be utilized in various experimental techniques to study the expression and localization of CPNE1 in biological samples.
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6 protocols using anti cpne1
1
Immunocytochemistry of CPNE1 and RACK1
2
Immunohistochemical Evaluation of CPNE1 in Prostate
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Immunohistochemical assay was conducted to examine CPNE1 expression in prostate samples according to the manufacturer’s protocol, as previously described [19 (link)]. Briefly, all prostate tissues on slides were deparaffinized and rehydrated. The slides were incubated with polyclonal rabbit anti-CPNE1 (1: 400 dilution, Abcam, Cambridge, UK) antibody. The CPNE1 expression was evaluated as described below. Briefly, a score represented the percentage of tumor cells with positive staining. Score 0 represented negative staining; score 1 represented positive tumor cell staining (<25%); score 2 represented positive tumor cell staining (25–50%); score 3 represented positive tumor cell staining (50–75%); and score 4 represented positive tumor cell staining (>75%). Then, a score was further assigned according to the staining intensity (score 0 indicated negative staining intensity; score 1 indicated weak staining intensity; score 2 indicated moderate staining intensity; and score 3 indicated strong staining intensity). According to scores of positive staining and staining intensity, a total score was assigned (Range: 0–7). The expression levels of CPNE1 were categorized into low expression (Scores: <4) or high expression (Scores: 4–7).
3
Immunofluorescence Assay for CPNE1 and TRAF2
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Immunofluorescence assay was conducted according to a previously described method [20 (link)]. Briefly, cells were incubated with polyclonal rabbit anti-CPNE1 (1: 400 dilution, Abcam, Cambridge, UK) or TRAF2 (1: 400 dilution, Bioworld Technology, Minneapolis, USA) antibodies, and subsequently were incubated with goat anti-rabbit IgG Rhodamine (TRITC) for 2 h at room temperature.
4
Co-immunoprecipitation and Western Blotting
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Western blotting (WB) was performed as previously described [33 (link)]. The antibodies used for the analysis were anti-CPNE1 (Abcam, Shanghai, China) and anti-NEDD4L (CST, UK). For co-immunoprecipitation experiments, 2 × 105 HEK293T cells were transfected with the required plasmids for 48 h. Cells were harvested with 1 ml of RIPA buffer. Equal amounts of protein were incubated with 2 μg of M2 anti-Flag affinity agarose at 4 °C for 4 h with end-over-end rotation. The protein–antibody complexes were collected by centrifugation and washed 3 times with RIPA buffer. Then, the precipitates were analyzed by Western blotting.
5
Protein Analysis in Colorectal Cancer
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Total protein extracts were prepared from CRC cells or tumor tissues using RIPA buffer and were quantified by the BCA protein assay kit (Thermofisher, Bridgewater Township, NJ, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were incubated with anti-CPNE1, anti-GLUT1, anti-HK2, anti-cleaved Caspase 3 (Abcam, Cambridge, MA), anti-AKT, anti-p-AKT, or anti-GAPDH antibodies (Santa Cruz, Santa Cruz, CA) at 4°C overnight. Bands were visualized using an ECL kit (BioVision, Exton, PA) with an LAS-400 image analyzer (FujiFilm Medical Systems, Stamford, CT).
6
Investigating CPNE1 and NEDD4L Interactions
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Western blotting (WB) was performed as previously described [29] . The antibodies used for the analysis were anti-CPNE1 (Abcam, Shanghai, China) and anti-NEDD4L (CST, UK). For co-immunoprecipitation experiments, 2×10 5 HEK293T cells were transfected with the required plasmids for 48 h. Cells were harvested with 1mlof RIPA buffer.
Equal amounts of protein were incubated with 2μg of M2 anti-Flag a nity agarose at 4 °C for 4 h with end-over-end rotation. The protein-antibody complexes were collected by centrifugation and washed 3 times with RIPA buffer. Then, the precipitates were analysed by Western blotting.
Equal amounts of protein were incubated with 2μg of M2 anti-Flag a nity agarose at 4 °C for 4 h with end-over-end rotation. The protein-antibody complexes were collected by centrifugation and washed 3 times with RIPA buffer. Then, the precipitates were analysed by Western blotting.
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