Fig. 2

ARP has short- and long-term hematologic effects in vivo. (A) ARP accumulates in the serum under stress. Top: Shown are Ponceau-stained lanes of gradient polyacrylamide gels (4–20%, Bio-Rad) loaded with 1) protein extract from COS cells transfected with AChE-R encoding plasmid and mixed with synthetic ARP, 2) recombinant human AChE-S (Sigma) mixed with synthetic ASP, 3) 2 µl serum from a mouse injected with saline, or 4) a mouse subjected to confined-swim stress 24 hr posttreatment. Positions of molecular weight markers are shown on the left. Bottom: The above shown gel was electroblotted and probed with affinity-purified rabbit antibodies elicited toward a recombinant GST-ARP fusion protein. Note labeling in the serum of a 67-KDa protein, consistent with the expected size of AChE-R; also note selective labeling of synthetic ARP (but not AChE-S or ASP) by this antibody and the appearance of a novel immunoreactive band of approximately 5 Kd in serum of the stressed mouse. (B) ARP facilitates stress-induced hematopoietic responses in vivo. Top panel: Shown are bone marrow smears immunostained with anti-GST-ARP antibodies. Note that AS1 reduced and ARP intensified immunolabeling (brown precipitates) and increased the number of small ARP-positive cells in stressed mice. Middle panel: Number of ARP-immunopositive cells in bone marrow from the noted groups of FVB/N mice; the graph represents an average of five different fields counted at 3 1000 magnification for bone marrow. Bottom panel: Peripheral white blood cell counts. Note that both bone marrow immunostaining and white cell counts revealed ARP-dependent elevations and AS1 suppression (n 5 12 mice per ARP and AS1 treated stress groups). Control, nonstressed FVB/N mice were injected intraperitoneally with normal saline (n 5 6) or ARP (n 5 4). (C) Clonogenic progenitor cultures. Bone marrow cells from the noted groups of mice were subjected to clonogenic progenitor assays as described in methods. Top: myeloid CFU-GEMM; bottom: CFU-MK. Asterisks denote statistical significance (p, 0.05, ANOVA). Increased numbers of CFU reflects increased numbers of viable progenitors able to give rise to each of the assayed cell lineages.