Blimp 1

Colon specimens were homogenized and the supernatant was extracted as described above. Protein concentration was determined by classical BCA (Beyotime, Nanjing China) protein assay. Equal amounts of total protein (60–80 μg) were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane using a Bio-Rad Western blotting apparatus. After blocking with 5% non-fat milk or bovine serum albumin, primary antibodies were added overnight at 4°C. The antibodies were anti-GAPDH (No. 160110) (1:3,000), BCL-6 (No. ab19011) (1:1,000), p-STAT3 (No. ab131103) (1:2,000), SAP (No. ab185810) (1:2,000), Blimp-1 (No. ab7888) (1:1,000), and STAT3 (No. ab119352) (1:2,000) (Abcam, Cambridge, MA, USA). The sections were incubated with a second antibody [Goat Anti-Rabbit lgG H&L (HRP)] (No. 150077) (1:2,000–1:4,000) (Abcam) for 1 h at 37°C. The labeled protein bands were scanned with an HP Scanjet 5500 (Hewlett Packard France, Les Ullis, France).

At the indicated time points, cells were lysed in Laemmli buffer. For cytoplasmic/nuclear protein samples, proteins were extracted using a cytoplasmic and nuclear extraction kit (Boster Bio), and protein concentration was determined by bicinchoninic acid assay (Boster Bio). Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. Proteins were detected by ECL (SuperSignal West Pico PLUS, Thermo Fisher Scientific) and visualized on a ChemiDoc (Bio-Rad) or film. Protein bands were quantitated using Image Lab 6.0.1 software (Bio-Rad) or ImageJ.
Abs used were p-AKT, AKT, p-ERK1/2, ERK1/2, p-FOXO1/3/4, FOXO1, FOXO3, p-JNK, JNK, p-p38, p38, MYC, RELA, p-SQSTM1 T269/S272, and SQSTM1 (CST); tubulin (Merck); H3 (Abcam), BLIMP-1 (R23) (22 (link)); and goat anti-mouse HRP and goat anti-rabbit HRP (Jackson ImmunoResearch).