Fig 2: A subset of AML patients and cell lines expresses detectable phosphorylated PKC. (A) AML cells from patient samples were subjected to Western blotting with an antibody that recognizes phosphorylated PKCa and PKCßII (pPKC). Actin was used a loading control. (B) HL60, K562, MFL2, MR2, and OCI-AML3 cells were harvested and subjected to Western blotting with an antibody that recognizes phosphorylated PKCa/ßII (pPKC) as well as total PKCa (PKCa). Actin was used a loading control.

Fig 3: The TANC2-PRKCA fusion is detected at the mRNA level but not the protein level in CRISPR-edited cells.A, schematic illustrating strategy to generate cells expressing the TANC2-PRKCA fusion via CRISPR/Cas9 gene editing. B, mRNA expression of the TANC2-PRKCA fusion and actin (ACTB) in the parental and CRISPR-edited clone analyzed by RT-PCR. Product indicated by black bar on schematic. Reactions analyzed on agarose gel, with each lane representing an individual replicate. C, TANC2 mRNA expression at the 5' (“N-Term”) and 3' (“C-Term”) ends in the parental and CRISPR-edited clone analyzed by RT-qPCR and normalized to actin (ACTB). Products indicated by black bars on schematic. Data represent means ± SEM from at least three independent experiments. **p < 0.01 (Student’s t-test). D, PRKCA mRNA expression at the 5' (“N-Term”) and 3' (“C-Term”) ends in the parental and CRISPR-edited clone analyzed by RT-qPCR and normalized to actin (ACTB). Products indicated by black bars on schematic. Data represent means ± SEM from at least three independent experiments. **p < 0.01, ***p < 0.001 (Student’s t-test). E, immunoblot analysis of lysates from parental and CRISPR-edited cells, probed for TANC2, PKCa, and a-tubulin. Arrow indicates predicted mobility of the fusion protein. Data are representative of three independent experiments.

Fig 4: TANC2-PRKCA-expressing cells exhibit reduced levels of cleaved caspase-3 and decreased apoptosis.A, immunoblot analysis of lysates from parental, CRISPR-edited TANC2-PKCa fusion, and heterozygous PKCa knockout cells, probed for caspase-3, cleaved caspase-3, PKCa, and ß-actin. Data are representative of three independent experiments. B, apoptosis assay performed on MDA-MB-231 parental (gray), CRISPR-edited TANC2-PKCa fusion (purple), or heterozygous PKCa knockout (orange) cells. Data represent luminescence upon binding of annexin V-luciferase to cell-surface-exposed phosphatidylserine as a function of time following addition of reagents. Graph depicts mean ± SEM from three independent experiments. **p < 0.01, ***p < 0.001 (Student’s t-test). Asterisk indicates significance between parental and fusion-expressing cells, and # indicates significance between parental and heterozygous PKCa knockout cells. C, apoptosis assay described in (B) was performed on MDA-MB-231 parental (gray), CRISPR-edited TANC2-PKCa fusion (purple), or heterozygous PKCa knockout (orange) cells treated with etoposide (12.5 µM) or vehicle (DMSO) for 24 h. Graph depicts mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test). Asterisk indicates significance between vehicle and etoposide-treated conditions for each cell line. # and % indicate significance between the parental and CRISPR-edited fusion or heterozygous PKCa knockout cells in basal and etoposide-treated conditions, respectively.

Fig 5: Detection of PICK1 and PKC signaling in brain tissues.The brain samples from WT and Tspan7-/- rats were, respectively, homogenized in RIPA lysis buffer, and the total proteins were obtained and analyzed by Western blot using primary antibodies of anti-p-ERK (4370; dilution of 1:1,000; CST), ERK (9102; dilution of 1:1,000; CST), p-PKCe (ab63387; dilution of 1:500; Abcam), PKC; (2683; dilution of 1:500; CST), p-PKCa (9375; dilution of 1:1,000; CST), PKCa (2056; dilution of 1:500; CST), PICK1 (10983-2-AP; dilution of 1:1,000; ProteinTech), NMDAR1 (ab17345; dilution of 1:500; Abcam), and secondary antibodies of HRP-labelled goat anti-mouse IgG, HRP-labelled goat anti-rabbit IgG. Proteins were visualized using a chemiluminescent detection system and analyzed using Quantity One software. (A) Phosphorylated and total ERK, PKCe, PKCa, PICK1, and NMDAR1 proteins in the brain tissues from WT and Tspan7-/- rats were detected with Western blot. (B, C, D, E, F) Phosphorylated and total ERK, PKCe, PKCa, PICK1, and NMDAR1 proteins in the brain tissues from WT and Tspan7-/- rats were quantified with ImageJ. (G) Body weights and birth rates were compared between WT and Tspan7-/- rats at 2, 3, 4, and 8 wk. The results showed that the PICK1, ERK, PKC, and NMDAR1 were not changed obviously in Tspan7-/- rats and suggesting PICK1 or ERK signal could be not the main pathway of Tspan7-/-. Data information: In (B, C, D, E, F), data were analyzed by two-tailed unpaired t tests. Error bars represent SEM; n.s, not significant.Source data are available for this figure.