Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: AKT Phosphorylates FAM13A and Promotes Its Degradation via CUL4A/DDB1/DCAF1 E3 Complex

doi: 10.1165/rcmb.2022-0362oc

Figure Lengend Snippet: Figure 1. AKT promotes FAM13A (family with sequence similarity 13 member A) degradation. (A) Protein levels of FAM13A were measured in NHBE cells and 16HBE cells after 3% cigarette smoke extract (CSE) treatment for 12 hours with recovery (CSER) or without recovery. Cells without CSE were used as a control. (B) FAM13A protein was detected and quantified in 16HBE cells pretreated with MG132 (10 mM for 3 h), followed by treatment with cycloheximide (CHX; 10 mg/ml) to inhibit new protein synthesis for indicated durations. (C) Normalized FAM13A protein levels from three replicates were fit to the curve of a nonlinear one-phase decay model for calculation of the half-life of FAM13A protein. (D) Protein levels of phosphorylated AKT, total AKT, and FAM13A were measured using IB in 16HBE cells at indicated time points. (E) Quantification of protein bands in D indicates correlation between FAM13A levels and AKT activation. (F) Detection of Flag-tagged FAM13A in 16HBE cells cotransfected with low or high amount of AKT1 or AKT2. (G) Detection of AKT activation and FAM13A in HEK 293T cells cotransfected with Flag-FAM13A and AKT followed by treatment with indicated inhibitors. ALLN = acetylleucyl-leucyl-norleucinal; NHBE = normal human bronchial epithelial; p-AKT = phosphorylated AKT.

Article Snippet: ELISA was performed using the Human Protein FAM13A ELISA Kit (orb1209526; Biorbyt) as described in the manual.

Techniques: Sequencing, Control, Activation Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: AKT Phosphorylates FAM13A and Promotes Its Degradation via CUL4A/DDB1/DCAF1 E3 Complex

doi: 10.1165/rcmb.2022-0362oc

Figure Lengend Snippet: Figure 2. AKT phosphorylates FAM13A to promote its degradation. (A) Detection of the ubiquitination of Flag-tagged FAM13A in 16HBE cells cotransfected with wild-type (WT) AKT or mutant AKTK179M with defective kinase activity after IP with Flag antibody. Cells were treated with MG132 before IP assay. (B) Detection of FAM13A protein in 16HBE cells cotransfected with siRNA targeting FAM13A or siRNA control and AKT followed by IP using the antibody recognizing phosphorylated AKT substrates. (C) Conserved AKT phosphorylation sites on FAM13A protein across species are indicated inside the orange frame. Highly conserved residues are highlighted in red. (D) Four potential phosphorylation sites on human FAM13A protein contain AKT substrate motif. (E and F) Predicted protein structural model simulating interaction between FAM13A

Article Snippet: ELISA was performed using the Human Protein FAM13A ELISA Kit (orb1209526; Biorbyt) as described in the manual.

Techniques: Ubiquitin Proteomics, Mutagenesis, Activity Assay, Control, Phospho-proteomics

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: AKT Phosphorylates FAM13A and Promotes Its Degradation via CUL4A/DDB1/DCAF1 E3 Complex

doi: 10.1165/rcmb.2022-0362oc

Figure Lengend Snippet: Figure 3. Phosphorylation of FAM13A at S312/S322 regulates the stability of FAM13A protein. (A) Measurements of the levels of endogenous FAM13A protein in 16HBE clonal lines edited using CRISPR/Cas9 targeting S312 site expressing WT or mutant FAM13A pretreated with MG132 (10 mM for 3 h) and then CHX (10 mg/ml) for indicated durations. (B) Detection of phosphorylated AKT and FAM13A in FAM13AWT, FAM13AS312A, or FAM13AS312D 16HBE clonal lines after CSER treatment. (C) Detection of FAM13A protein in prestarved 16HBE clonal lines (FAM13AWT, FAM13AS312A, or FAM13AS312D), followed by INS (2.5 mg/ml) treatment for indicated durations. (D) S312-phosphorylated FAM13A was assessed using immunoblots in 16HBE clonal lines expressing FAM13AWT or FAM13AS312A under indicated conditions. (E) Ubiquitination of FAM13A was detected using IP in 16HBE cells transfected with Flag-tagged WT FAM13A or various mutants (FAM13AS312A or FAM13AS312D) and treated with CSER. In D and E, cells were pretreated with MG132 to prevent protein degradation. Cas9 = CRISPR associated protein 9; CHX = cycloheximide; INS = insulin.

Article Snippet: ELISA was performed using the Human Protein FAM13A ELISA Kit (orb1209526; Biorbyt) as described in the manual.

Techniques: Phospho-proteomics, CRISPR, Expressing, Mutagenesis, Western Blot, Ubiquitin Proteomics, Transfection

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: AKT Phosphorylates FAM13A and Promotes Its Degradation via CUL4A/DDB1/DCAF1 E3 Complex

doi: 10.1165/rcmb.2022-0362oc

Figure Lengend Snippet: Figure 4. CULLIN4A E3 ligase complex contributes to the degradation of FAM13A. (A) Myc-tagged CULLIN family proteins interact with overexpressed FAM13A as assessed using co-IP assays in HEK 293T cells. Immunoprecipitated FAM13A:CULLIN protein ratios were calculated on the basis of band densitometry as shown. (B) A model diagram showing FAM13A binding to CULLIN4A (CUL4A) E3 ligase complex. (C) Detection of FAM13A in 16HBE cells cotransfected with AKT and siRNA targeting indicated CULLIN4A complex component or scramble siRNA. (D) Ubiquitination of Flag-tagged FAM13A was detected in 16HBE cells cotransfected with HA–tagged ubiquitin, indicated siRNA, and Flag-FAM13A by IP with Flag beads. Cells were pretreated with MG132 (10 mM for 3 h) before IP experiment. (E) Interaction of CULLIN4A complex with endogenous FAM13A in 16HBE clonal lines (FAM13AWT, FAM13AS312A, or FAM13AS312D) detected using IP. DCAF1 = DDB1 and CUL4 associated factor 1; DDB1 = damage specific DNA binding protein 1; HA = hemagglutinin; RBX1 = Ring-Box 1; Ub = ubiquitin.

Article Snippet: ELISA was performed using the Human Protein FAM13A ELISA Kit (orb1209526; Biorbyt) as described in the manual.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Binding Assay, Ubiquitin Proteomics

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: AKT Phosphorylates FAM13A and Promotes Its Degradation via CUL4A/DDB1/DCAF1 E3 Complex

doi: 10.1165/rcmb.2022-0362oc

Figure Lengend Snippet: Figure 5. S322-Fam13a was phosphorylated in mouse lung injury models in vivo. (A) Schematic illustration of the influenza-induced lung injury/repair mouse model. (B) Indicated protein levels of Fam13a, S322-phosphorylated Fam13a, and activated Akt were measured in lung tissues from mice after influenza infection. (C) Phosphorylated Akt (S473):total Akt ratio, phosphorylated Fam13a (S322):total Fam13a ratio, and normalized Fam13a protein levels were quantified in B across different time points. (D) Correlations between Akt phosphorylation and Fam13a total protein or Fam13a phosphorylation at S322 were analyzed. (E) Diagram showing the design of the naphthalene-induced

Article Snippet: ELISA was performed using the Human Protein FAM13A ELISA Kit (orb1209526; Biorbyt) as described in the manual.

Techniques: In Vivo, Infection, Phospho-proteomics

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: AKT Phosphorylates FAM13A and Promotes Its Degradation via CUL4A/DDB1/DCAF1 E3 Complex

doi: 10.1165/rcmb.2022-0362oc

Figure Lengend Snippet: Figure 6. FAM13A suppresses cell growth in vitro and in vivo. (A) Cell growth curve in 16HBE cells stably infected with lentiviral shRNA targeting FAM13A (FAM13Ash). Cells infected with nontargeting shRNA were used as controls (STDsh). Mean 6 SD values are from triplicate wells in one representative repeat from three biological repeats. (B) Cell growth curves in stable 16HBE clonal lines (FAM13AWT, FAM13AS312A, or FAM13AS312D). (C) Immunostaining with club cell 10 kD (CC10) (red), SP-C (surfactant protein C) (green), BrdU (yellow), and DAPI (blue), indicating club cells, alveolar type II (ATII) cells, proliferating cells, and cell nucleus, respectively. Scale bars, 25 mm. (D) Quantification of proliferating ATII cells (BrdU1SP-C1/SP-C1) in Fam13a1/1 and Fam13a2/2 mice (n = 4) after influenza infection. Each dot indicates the quantification result of a randomly chosen area (0.5 3 0.4 mm) on the lung specimen. Yellow arrows indicate BrdU1SP-C1 cells. White dashed lines indicate airway boundaries. (E) Immunofluorescence staining in lung specimens from Fam13a1/1 and Fam13a2/2 mice after naphthalene treatment with CC10 (red), SP-C (green), BrdU (yellow), and DAPI (blue), indicating club cells, ATII cells, proliferating cells, and cell nucleus, respectively. Yellow arrowheads indicate BrdU1CC101 cells. White dashed lines indicate airway basal membrane. Scale bars, 25 mm. (F) Quantification on the number of CC101 cells per airway. (G) Proliferating CC101 cells (i.e., percentage of BrdU1CC101 cells/CC101 in

Article Snippet: ELISA was performed using the Human Protein FAM13A ELISA Kit (orb1209526; Biorbyt) as described in the manual.

Techniques: In Vitro, In Vivo, Stable Transfection, Infection, shRNA, Immunostaining, Immunofluorescence, Staining, Membrane