Fig 2: Comparison of p-FMS/FMS between CSF-1 stimulated and unstimulated PBMCs. In order to compare the two assays, fold difference assessed in figure 4 was averaged between the donors (N=5) using the IP-Western and the IP-MSD assay and plotted as shown, with the left y-axis capturing the IP-Western data and the right y-axis capturing the IP-MSD data.

Fig 3: Dilutional linearity of PBMC lysates in IP-MSD assay. PBMC lysates were divided into two groups- CSF-1 stimulated (+) and non-stimulated (-). FMS and p-FMS were measured for both the samples using the MSD assay. The samples were sequentially diluted 50% in lysis buffer and each of the diluted samples analyzed in both the FMS and p-FMS assays. The dotted and solid vertical lines display the minimum PBMC lysate volumes (μL) needed for the p-FMS and FMS assays, respectively.

Fig 4: Comparison of p-FMS/FMS ratios between CSF-1 stimulated and unstimulated samples from whole blood. 3A. IP-Western; 3B. IP-MSD assay; 3C. Comparison of the average values of the five donors, for both conditions, in both assays. Fold difference reflects the effect of CSF-1. PBMCs from whole blood were collected, and samples were divided into two categories: containing DMSO (+) and without DMSO (-). Each sample was further sub-divided into two- stimulated and unstimulated- and for each sample p-FMS and FMS were both assayed. Ratio between p-FMS and FMS was determined for each sample and ratios of p-FMS/FMS between stimulated and unstimulated samples were assessed to indicate the fold difference.

Fig 5: MTX modulates the expression of MAF, MAFB, and CSF1R in M-CSF monocyte-derived macrophages. a Immunoblot analysis of MAF and MAFB by M-MØ and MTX-M-MØ. b Relative level of expression of the indicated genes as determined by RNA-sequencing on M-MØ and MTX-M-MØ (GSE186151). c Left, Immunoblot analysis of CSF1R by M-MØ and MTX-M-MØ. Right, immunoblot analysis of CSF1R by M-MØ, MTX-M-MØ, or MTX-M-MØ exposed to FA. GAPDH protein levels were determined as protein loading control. d Immunoblot analysis of pCSF1R (left), pERK1/2, and total ERK (right) by M-MØ and MTX-M-MØ challenged with M-CSF for the indicated time points. For a, c, d, two–three independent donors are shown. Vinculin, tubulin, or GAPDH protein levels were determined as protein loading control. Arrowheads indicate the protein of interest.