Fig 1: Ser-322 is important for subcellular distribution of Fam13a and its interaction with 14-3-3. (A) Representative immunofluorescence images, showing the subcellular distribution of Fam13a and S322A in NIH3T3 cells treated with DMSO (control) or OA. Bar graph indicates the percentage of cells that exhibit nuclear, cytoplasmic, or homogeneous subcellular distribution. (B) A GST pull down, showing that Ser-322 is important for the binding between Fam13a and 14-3-3 and the effect of OA on this interaction. Fam13a- and S322A-expressing NIH3T3 cells were treated with DMSO (control) or OA. Cell lysates were used in a GST pull-down assay. Interaction between Fam13a and 14-3-3 was markedly enhanced in OA-treated cells. In contrast, S322A mutant showed weaker interaction with 14-3-3 in OA-treated cells. (C) Western blot, showing the specificity of the anti-phosphoS322 (pS322) antibody. Note that the anti-pS322 antibody strongly reacted with Fam13a. It did not react with ?315–329 but recognized S322A weakly. (D) Western blot, showing Ser-322 phosphorylation in NIH3T3 cells treated with DMSO (control), OA, wortmannin, or OA plus wortmannin. pS322 level was increased by OA treatment. The effect of OA on pS322 was reduced by wortmannin treatment. (E) Western blot, showing that overexpression of B56ß, B56?, B56d, and B56e decreased Ser-322 phosphorylation. All B56 expression constructs were FLAG tagged.
Fig 2: Interaction between Fam13a and 14-3-3. (A) GST pull down, showing that Fam13a interacted with bacterially expressed GST–14-3-3? and GST–14-3-3e but not GST. (B) Western blot, showing coimmunoprecipitation of myc-Fam13a and FLAG–14-3-3?. FLAG–14-3-3? was detected when myc-Fam13a was immunoprecipitated (middle). Conversely, myc-Fam13a was detected when FLAG-14-3-3? was immunoprecipitated (right). (C) GST pull-down assay, showing that GST–14-3-3 interacts with the full-length Fam13a and some Fam13a deletion constructs (1–609, 1–513, 1–393, and 1–341). GST-14-3-3 failed to pull down 1–158. (D) Western blot, showing that truncation of residues 315–329 decreased the binding between GST–14-3-3 and Fam13a.
Fig 3: Loss of Fam13a minimally affects adiposity, diet-induced obesity and metabolic homeostasis in vivo. a FAM13A protein expression in various murine tissues. b Fat and lean masses presented as % of body weight. c Tissue masses (mg) as normalized to BW (g). n = 6–7/group. d Representative H&E images of eWAT, sWAT, BAT and liver. Scale bar, 100 µm. e Representative western blot of FAM13A and adipose markers PPAR? and PLIN1 in eWAT, n = 4/group. f, g Glucose tolerance test (f) and (g) insulin tolerance test. Chow fed 14–15 week old male Fam13a+/+ (+/+) and Fam13a-/- (-/-) mice were used for experiments performed in a. g n = 6–7/group. h Tissue masses (mg) as normalized to BW (g) after 20 weeks of HFD feeding. n = 5–7/group. i, j Glucose tolerance test (i) and insulin tolerance test (j) on mice fed with HFD for 11 weeks and 13 weeks respectively (n = 6–7 per group)
Fig 4: Morphological observation of normal liver tissue (a) and cirrhotic tissue (b), and the expression of FAM13A in normal liver tissue (c) and cirrhotic liver tissue (d)
Fig 5: Identification of NLSs in Fam13a. (A) Schematic diagram of wild-type Fam13a and NLS mutants (RR340AA, RR531AA, and RR340;531AA). (B) Representative immunofluorescence images showing the nuclear localization of wild-type Fam13a and mislocalization of NLS mutants in NIH3T3 cells. Bar graphs show percentage of cells that exhibit nuclear, cytoplasmic, or homogeneous subcellular distribution. R, arginine; A, alanine.
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