Phospho atf 2 thr71

Cells were lysed in Laemmli buffer and analyzed for total protein concentration^{56 (link)}. Fifteen microgram of total protein from each cell lysate was separated by SDS-PAGE and transferred onto a PVDF membrane (Invitrogen). The membranes were probed with primary antibodies. Primary antibodies: RBFOX2 (1:4000, Sigma), SRSF1 (1:200, mAb AK96 culture supernatant^{57 (link)}), SRSF5 (1:3000, Sigma), SRSF6 (1:500, mAb 8-1-28 culture supernatant), OctA (Flag tag) (1:500, Santa Cruz), hnRNPM (1:500, Novusbio), PTBP1 (1:10,000, Abcam), T7 (1:5000 Novagen), phospho-p38 (Thr180/Tyr182) (1:1000 cell signaling), p38 (1:1000 Santa Cruz), phospho- ATF2 (Thr71) (1:1000 Cell Signaling), ATF2 (1:1000 abcam), β tubulin I + II (1:1000, Sigma), β-actin (1:200, Santa Cruz), GAPDH (1:1000, Santa Cruz), Secondary antibodies: HRP-conjugated goat anti-mouse, goat anti-rabbit, or donkey anti-goat IgG (H + L) (1:10,000, Jackson Laboratories).

Tan I (purity ≥98%) was purchased from Yuanye Bio-Technology (China) and its structure is shown in Figure 1A. The drug was dissolved in DMSO and stored at -20°C. RPMI 1640 medium was obtained from Cellmax (China) and the fetal bovine serum (FBS) was from Sijiqing (China). MTT assay kit and DAPI-staining kit were obtained from Beyotime Biotechnology (China) and Annexin V-FITC+PI double-staining kit was purchased from BD Biosciences (USA). RNA-Quick Purification Kit was purchased from Yishan Biotechnology (China) and both the mRNA reverse transcription kit and RT-PCR kit were obtained from Yeasen Biotechnology (China). BCA Protein Assay Kit was from Beyotime Biotechnology and the antibodies used in this study were purchased from Cell Signaling Technology (USA): cleaved caspase 3 (Cat 9664), cleaved caspase 9 (Cat 9505), cleaved PARP (Cat 5625), PUMA (Cat 12450P), Bcl-2 (Cat 3498), phospho-SAPK/JNK (Thr183/Tyr185) (Cat 4668), JNK (Cat 9252), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cat 4370), phospho-ATF-2 (Thr71) (Cat 5112), and β-actin (Cat 3700). Additionally, ERK1/2 (Cat AF0155) was from Affinity Biosciences (USA) and secondary antibodies were obtained from Santa Cruz Biotechnology (USA).