Proteins were extracted from ventricular tissue using lysis buffer and measured using a BCA assay kit (Beyotime). SDS-PAGE was applied to separate the total protein (50 µg), which was then transferred to PVDF membranes. The membranes blocked and incubated with primary and secondary antibodies against POLR1D (1:400, ab243591, rabbit polyclonal, Abcam); Cleaved caspase-3 (1:500, ab32042, rabbit monoclonal, Abcam); E-cadherin (1:500, 20874-1-AP, rabbit polyclonal, Proteintech); N-cadherin (1:500, 22018-1-AP, rabbit polyclonal, Proteintech); p-PI3K (1:500, #4228, rabbit polyclonal, Cell Signaling Technology); PI3K (1:500, R22768, rabbit polyclonal, ZenBio); p-AKT (1:500, #4060S, rabbit polyclonal, Cell Signaling Technology); AKT (1:500, #4691, rabbit polyclonal, Cell Signaling Technology) and GAPDH (1:2000, sc-47,724, mouse monoclonal, Santa Cruz) were using the 5% skimmed milk. Then, membranes were washed with a solution of Tris-buffered saline and Tween-20 before being treated with a secondary antibody conjugated to horseradish peroxidase for two hours at room temperature. For repeated use of PVDF membrane, antibody stripping solution (WB6500, NCM Biotech, Suzhou, China) was used. ECL reagents were used to identify protein bands (Amersham Biosciences, UK). ImageJ software was used to examine the protein bands, and GAPDH was employed as an internal control.
Ab243591
Manufactured by Abcam
Sourced in United Kingdom
Ab243591 is a laboratory product manufactured by Abcam. It is a piece of lab equipment used for scientific research purposes. The core function of this product is to facilitate specific laboratory procedures, but no further details on its intended use can be provided in an unbiased and factual manner.
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Lab products found in correlation
2 protocols using ab243591
1
Western Blot Analysis of Protein Expression
2
Immunohistochemical Analysis of FAT4 in CRC
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FFPE samples (CRC and adjacent tissues) were sectioned into 4µm thick, and prepared for immunohistochemistry staining. The sections were incubated with rabbit polyclonal anti-FAT4 antibody (ab243591, 1:200, Abcam, UK) at 4 °C overnight. After washing the sections by PBS, the sections were then incubated with goat anti-rabbit secondary antibody (ab205718, 1:2,000, Abcam, UK) at room temperature for 30 min. The peroxidase reactivity was measured by 3,3’-diaminobenzidine (DAB) (7411-49-6, Suzhou Yacoo Chemical Reagent Co., Ltd, China) and counterstained by haematoxylin. The IHC evaluation was conducted according to the description of Kusinska (16 (link)). The score grade of IHC intensity was classified as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Based on the scores, positive cells were accordingly assigned into four groups, namely, 0 (negative), 1(less than 10%), 2 (from 10% to 50%), and 3 (more than 50%) groups. The score of IHC intensity multiplied by the percentage of positive cells was expressed as the final score. If the final score was lower than or equal to 1, it was considered to be negative, while the score ranging from 2 to 9 was positive.
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