Ab198288

Western blotting was performed as previously described.21 (link) The following primary antibodies were used: APOC1 (1:1000, ab198288), cyclin B1 (1:5000, ab32053), cyclin D1 (1:5000, ab134175), caspase-9 (1:1000, ab202068), BCL-2 (1:500, ab32124), ERK1/2 (1:500, ab196883), phospho-ERK1/2 (1:500, ab65142), and GADPH (1:2500, ab9485) were purchased from Abcam Inc. Phospho-p38 (1:1000, #9211), p38 (1:1000, #9212), phospho-JNK (1:1000, #9251) and JNK (1:1000, #9252) were purchased from Cell Signaling Technology (Boston, MA, USA). Goat anti-rabbit IgG H&L (1:2000, ab6721, Abcam) was used as a second antibody.

SAOS-2 cells were lysed in RIPA buffer (Protech Technology Enterprise Co., Ltd.) including protease inhibitors at 4˚C to obtain the protein. Subsequently, samples (250 µl) were incubated overnight at 4˚C with IgG (1:50; cat. no. ab172730; Abcam), anti-APOC1 (1:40; ab198288; Abcam) or anti-MTCH2 antibodies (1:50; 16888-1-AP; Proteintech). A total of 30 µl protein A/G magnetic beads (MilliporeSigma) was added and incubated overnight at 4˚C. The beads were washed with PBS and immunoprecipitants were assessed via western blotting.

The following procedures were performed by classical biotin–streptavidin–peroxidase IHC staining protocols according to previously described methods.19 (link) Briefly, the protein expression of APOC1 was detected by anti-APOC1 (1:200, ab198288, Abcam Inc, Cambridge, MA, USA) antibody and a goat anti-mouse/rabbit secondary antibody (Gene Tech Co Ltd, GTVisionTM III Detection System/Mo&Rb, Shanghai, China). To estimate the score for the 140 cases of CRC paraffin-embedded samples, five high-power fields were chosen randomly for the assessment of APOC1 expression in a double-blind procedure. The sample scores were based on both the intensity of staining and the proportion of positively stained tumor tissue. The immunostaining results were scored based on the following system: for staining intensity scoring: 0 (negative), 1 (weak), 2 (moderate), 3 (strong); for staining area scoring: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). The staining index was calculated as the product of the proportion of positive tissue × the staining intensity score (range from 0 to 12). An optimal cutoff value was identified as follows: a staining index score ≥6 was considered as tumors with high APOC1 expression and ≤4 was considered as tumors with low expression of APOC1.20 (link)