Fig 2: LAMA5 mutant chondrocytes and LAMA5Hyp/Hyp Hela cells dysregulate SRC kinase expression levels. (a) Chondrocytes from R03–206 G showed decreased levels of p-SRC family. (b) A similar decrease in p-SRC family, as well as in FYN, was observed in HeLa LAMA5Hyp/Hyp cells. (c) Quantitation of the decreased FYN levels in LAMA5Hyp/Hyp HeLa cells shown in b. (d-j) PYK2 localizes similarly to LAMA5 around blood vessels in cartilage, ligaments and bone as well as articular cartilage growth plate and periosteum/perichondrium in human skeletal tissue. (k-q) FYN shows a very similar pattern with the exception of proliferative and hypertrophic growth plate chondrocytes, where it was not seen. Bars represent 50um.

Fig 3: Optimization of Sleeping Beauty stable cell lines and antibody staining. (A and D) ARPE19 cells stably expressing eGFP-clathrin were treated with siRNA targeting Fyn, Epsin1, or nontargeting (control [con.]) siRNA. After transfection, cells were fixed and stained with antibodies recognizing Fyn (A) or Epsin (D), followed by imaging by TIRFM and quantification of signaling intensity per cell. Shown are representative TIRFM images (left) and the measurements of Fyn (A) or Epsin (D) signal intensity (right). Scale bar = 20 μm. Both Fyn and Epsin1 antibodies are highly specific. (B and C) Stable cell lines harboring a transgene for doxycycline-inducible expression of eGFP-TOM1L1 or Fyn-iRFP713 were treated with doxycycline as indicated for 24 h. Whole-cell lysates were resolved by Western blotting and probed with anti-TOM1L1 (B) or anti-Fyn antibodies (C). From these experiments, we optimized induction at 150 ng/ml doxycycline to obtain near-endogenous expression levels of eGFP-TOM1L1 and Fyn-iRFP713. Molecular weight markers (kD) are shown on the right. Source data are available for this figure: SourceData FS2. R.U., relative unit.

Fig 4: 6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances NADP+ binding affinity of 6PGD. a U87/epidermal growth factor receptor (EGFR) cells stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml-1) for 30 min. Cell lysates were incubated with Cibacron blue beads mimicking NADP+ for a pulldown assay. b In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without active recombinant Fyn. ITC assays were performed with pulldown 6PGD variants (0.05 mM) and NADP+ (1 mM) (top panel). His peptides were used as negative control. Statistical analyses of Kd values of 6PGD variants for NADP+ were presented (bottom panel). c, d In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without recombinant active Fyn. Km (c) and kcat (d) of 6PGD variants were determined. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. b–d Data represent the mean ± SD of three independent experiments. Student’s t-test (unpaired, two tailed), **p < 0.01; n.s. not significant. Source data are provided as a Source Data file

Fig 5: Fyn-CDK5 pathway is activated in NP 60 min after exposure to media from microglia overexpressing Sema 3A. A Cartoon summarizing the proposed Sema 3A downstream pathway in NP exposed to Sema 3A media for 60 min. Representative WB analyses of Fyn pTyr420 and Fyn B, CDK5 pTyr15 C and CDK5/p35 D as well as of the correspondent β actin. OD analysis is reported on the right. Fyn, p35/CDK5 and CDK5 pTyr15 OD values were normalized to β actin and expressed as a percentage of Ctrl. Fyn pTyr420 levels were calculated as a ratio of Fyn pTyr420 relative to the corresponding Fyn OD values (Fyn pTyr420/Fyn). N = 3. One-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; **P < 0.01