Fig 2: Study B: Western blot and PCR results.A+B: Identification of the Epo-R in skeletal muscle tissue in the non-stimulated state in all 10 subjects with the C20 (A) and M20 (B) antibodies. Positive control is the k-562 cells. M is the molecular marker. C: Activation of the Epo-R by phosphorylation evaluated by western blotting. The samples from the same subject in the stimulated and un-stimulated situation are loaded next to each other D: Phosphorylation of Epo-R, STAT5, p38-MAPK, Akt, p70S6 kinase, Lyn, and IKK, all normalized to the levels of the given protein. The mRNA levels of SOCS3 and IGF-I are normalized to beta-actin mRNA content. Black bars: placebo, white bars: rHuEpo. All results are from biopsies taken 1 h post treatment with either placebo or rHuEpo.

Fig 3: Loss of GADD34 increased cell activation signaling induced by LPS with Tyr/Cys-deprivation.(a). GADD34-deficient or control RAW 264.7 cells were treated with LPS (1 µg/mL) with Tyr/Cys-deprivation for the indicated times (0–24 h). Cell lysates were immunoblotted with the indicated antibodies. Graph shows the relative expression as means ± SE of three independent experiments. (b). Cell lysates of GADD34-deficient or control RAW 264.7 cells were subjected to immunoprecipitation (IP) using anti-GADD34 antibody. Immunoprecipitates were immunoblotted (IB) with anti-Lyn and anti-GADD34 antibodies. Data are representative of three independent experiments. The original immunoblots are presented in Supplementary Figure 4. *p < 0.05, **p < 0.01, *** p < 0.001.

Fig 4: Kinases Lyn and Syk selectively regulate Ras isoform’s activation through Ras-GEFs. a CD40 activates the upstream kinases Lyn and Syk in a dose-dependent manner, with lower doses activating Syk and higher doses activating Lyn. b Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Syk- or Lyn-specific siRNA transfected, anti-CD40 antibody (3 µg/ml) treated P388D1 cells. c Immunoblot analyses of phospho-tyrosine (p-tyr-Vav)), and Vav translocated Sos-1/2 (Tr-Sos-1/2), translocated Ras-GRP (Tr-RasGRP), CD71, syk, lyn and ß-actin in the lysates of untreated or Syk siRNA or Lyn siRNA or anti-CD40 antibody (3 µg/ml) treated P388D1 cells. d Immunoblot analysis of phospho-tyrosine (p-tyr Vav) and Vav translocated Sos-1/2, translocated Ras GRP, CD71, Lyn and phospho-Lyn, Syk and phospho-Syk (p-syk) in the lysates of untreated or Syk inhibitor (Syk Inh, 3 µM; Calbiochem, San Diego, CA) or PP-1 (340 nM; BIOMOL International, PA) treated and/or anti-CD40 antibody (3 µg/ml) treated macrophages. e Analysis of the association of Syk or Lyn with Sos-1/2, Vav, and Ras-GRP in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 µg/ml, 3 µg/ml and 6 µg/ml) treated macrophages.

Fig 5: siRNA knockdown of known regulators modulates osteoblast differentiation in primary calvarial osteoblasts. (A) Scheme showing consequence of siRNA knockdown of regulators on osteoblast differentiation. (B,E) Representative microscopic images of DRAQ5 and ELF 97 stained cells after siRNA treatment against Sp7 and Runx2 using DRAQ5 (red) and ELF 97 (green) for nuclear and ALP staining, respectively. (C,F) Percentage of cell number after Runx2 and Sp7 siRNA knockdown, respectively. (D,G) Percent cellular ALP activity after Runx2 and Sp7 siRNA knockdown, respectively. (H,K) Representative microscopic images of DRAQ5 and ELF 97 stained cells after siRNA treatment against Lyn and Src, using DRAQ5 (red) and ELF 97 (green) for nuclear and ALP staining, respectively. (I,L) Percentage cell number after Lyn and Src siRNA knockdown respectively. (J,M) Percent cellular ALP activity after Lyn and Src siRNA knockdown respectively. Data are expressed as mean ± SEM (n = 3). Scale bar: 200 µM. *p < 0.05, **p < 0.01, ***p < 0.001.