Fig 2: ATG9A induces Her2 protein degradation via c-Cbl E3 ubiquitin ligase(A) Immunoprecipitation of ATG9A and (B) Her2 followed by western blotting of indicated antibodies in BT474-TR and BT474 cells. IgG antibodies were used as controls. (C) Immunoprecipitation of Her2 in BT474-TR and (D) BT474 cells transfected with vector and ATG9A-encoding plasmid in the presence of trastuzumab (20 µg/ml) for 48 hours, followed by western blotting of indicated antibodies. (E) Western blotting for Her2, ATG9A, pAKT and c-Cbl upon sic-Cbl with or without ATG9A overexpression in the presence or absence of trastuzumab (20 µg/ml) in BT474-TR. Protein levels were analysed and quantified using Image J software. Her2 bands were normalised to a-tubulin. (F) BT474 and BT474-TR cell proliferation was analysed after siRNA silencing against c-Cbl (or a non-targeting siRNA (siCTL)), followed by overexpression of ATG9A (or control vector).

Fig 3: c-Cbl induces K63 Her2 polyUb redirecting for lysosomal targeted degradation(A) Effect of ATG9A on phosphorylation of c-Cbl in BT474-TR. Cells were transfected with vector and ATG9A-encoding plasmid in the presence of trastuzumab (20 µg/ml). Protein levels for ATG9A, c-Cbl total and phosphorylation sites Tyr 731 and 774, Her2 total and phosphorylated Tyr 1112 were quantified by Image J software. Bands were normalised to a-tubulin. (B) Immunoprecipitation of Her2 followed by western blotting for K63 and Her2 in BT474-TR cells transfected with siRNA against c-Cbl or siRNA, upon overexpressing ATG9A or control vector. IgG was used as control. (C) Immunofluorescence staining for LAMP1 and Her2 in BT474-TR cells upon transfection with vector or ATG9A-encoding plasmid in the presence of trastuzumab (20 µg/ml). Percentage of Her2/LAMP1 co-localisation was quantified using LEICA LAS AF lite software. (D) Immunofluorescence staining for GFP-ATG9A and LAMP1 in BT474 and BT474-TR transfected with ATG9A-GFP encoding plasmid in the presence of trastuzmab (20 µg/ml). Data is representative of three experiments. *P < 0.05, unpaired Student's t test.

Fig 4: Differential expression of c-Cbl tunes LynA protein levels and SFK-mediated downstream signaling in macrophages and mast cells.(A) Flow cytometry showing surface expression of the mast-cell markers FceR1a and c-Kit (CD117) in CskAS bone-marrow-derived mast cells (red) compared to peritoneal B cells (gray). (B) Immunoblots of CskAS mast cells and BMDMs (m?) after resting or IFN-? priming, showing the levels of LynA, c-Cbl, Cbl-b, and pErk1/2 during 3-IB-PP1 treatment; total Erk1/2 is shown as a loading control. (C–F) Quantification of c-Cbl, Cbl-b, LynA, and pErk in mast cells (red), IFN-?-primed m?s (dark teal), and rested m?s (light teal). (C) Quantification of steady-state c-Cbl and Cbl-b protein in mast cells and rested m?s, corrected for total protein content (TPS) and reported relative to levels in primed m?s. SEM, n = 3. Sig. from one-way ANOVA with Sidak’s multiple comparison test: [mast vs. rested m?s] **p=0.0035. Others pairs ns. (D–E) Quantification of LynA in mast cells and m?s during 3-IB-PP1 treatment, corrected for total protein content (TPS) and reported relative to the steady-state level of LynA in each cell type (D) or relative to the steady-state level of LynA in rested m?s (E). SEM, n = 3 for mast cells and primed m?s, n = 4 for rested m?s. Sig. from ANOVA2-Tukey: [mast vs. rested m?s, light teal asterisks], [mast vs. primed m?s, dark teal asterisks]; ***p=0.0004–0.0008, **p=0.0025–0.0088, * P=0.0134–0.0466. Other pairs ns. (F) Quantification of pErk1/2 induction during 3-IB-PP1 treatment, corrected for total Erk1/2 expression and reported relative to the steady-state level of pErk1/2 in rested m?s. SEM, n = 3 for mast cells, n = 4 for primed m?s, n = 5 for rested m?s. Sig. from ANOVA2-Sidak: [mast vs. rested m?s, asterisk]; *p=0.0330. Other pairs ns. (G) Immunoblots showing steady-state expression of c-Cbl in CskAS mast cells transfected in the presence or absence of Cbl-specific saRNA; Erk1/2 is shown as a loading control. (H) Immunoblots showing levels of LynA protein, pSyk, and pErk1/2 during 3-IB-PP1 treatment of Cbl-saRNA-transfected or mock-transfected mast cells. (I–J) Quantification of LynA and pErk in mast cells with (pink) and without (red) saRNA-induced upregulation of c-Cbl protein. SEM, n = 3. (I) Quantification of LynA protein during 3-IB-PP1 treatment, corrected for total protein content (TPS) and reported relative to the steady-state level for each transfection condition. Sig. from mixed-effects ANOVA analysis: *p=0.0229. (J) Quantification of pErk1/2 induction during 3-IB-PP1 treatment in saRNA-treated and mock-treated mast cells, corrected for total Erk1/2 expression and reported relative to the steady-state level for each condition. SEM, n = 3 for mast cells. Sig. from ANOVA2-Sidak: **p=0.0045. Refer to Figure 9—source data 1.10.7554/eLife.46043.041Figure 9—source data 1.Comparison of mast cells and macrophages.

Fig 5: Expression levels of Lyn, Cbl, and Cblb are regulated differentially in myeloid cells.Mouse RNAseq data derived from the Immunological Genome Project (ImmGen, http://rstats.immgen.org/Skyline/skyline.html, Heng and Painter, 2008; Dwyer et al., 2016). Lyn, Cbl, and Cblb mRNA expression levels are shown for macrophages, mast cells, neutrophils, dendritic cells, and monocytes. Refer to Figure 8—source data 1.10.7554/eLife.46043.039Figure 8—source data 1.Expression data from Immgen.