1. 1. FXM phospho-specific antibody mix 1 Introduction Summary Gene perturbation by RNAi: See posters from the Molecular Biology lab and the Macrophage Biology labs. Ligand treatment: 1x10 6 RAW 264.7 cells were plated in each well in a 6-well plate, cultured overnight, and serum starved for 1 hr before ligand treatment. Cells were treated with 100 nM C5a, 25  M UDP for C5a or UDP receptor activation. For Fc   receptor cross-linking, unless indicated otherwise, cells were treated with 5  g/ml IgG2a for 30 min, then with 44  g/ml F(ab)’ 2. Cells were treated for 1, 3, 10 or 30 min at 37 degree before harvest. Western Blot: Preparation of RAW 264.7 lysates for western blot analysis: PP00000168 Protein transfer: PP00000005 Western blot analysis for RAW 264.7: PP00000181 We completed an extensive screen for antibodies to use in our analyses of phosphorylatioin-state changes in response to stimulation by C5a, UDP and Fc  ligation. Ligand-specific patterns of protein phosphorylation were observed in the RAW cell extracts. Nevertheless, the detected changes in phosphorylation-state for each ligand were limited. For example, of over forty phospho-proteins analyzed in our initial screen of antibodies, only changes in phospho-Akt (S473) immunoreactivity were reliably detected in C5a or Fc  receptor activated RAW cells. Based on these results and evaluations previously done in the antibody lab, we have selected about 10 phospho-specific antibodies to form three tentative FXM phospho-specific antibody mixes. Among which, the mix 1 is most promising. We have begun to examine FXM ligands-induced protein phosphorylation changes in cell lines where particular gene expression is knock-down via RNAi. Preliminary results from these studies are reported here. Acknowledgements Protein Phosphorylation Studies in RNAi-mediated Gene Knock- down Cells Yan G. Ni, Lily Jiang, Tamara Roach, Robert Rebres, Iain Fraser and Joelle Zavzavadjian Scientific Staff of the AfCS Cell Preparation and Analysis Laboratory Methods FXM Antibody Mix 1 Appare nt MWSource FAK (Y925)125CST3284 Pyk2 (Y402)116CST 3291 p-p90RSK (S380)90CST 9341 p-Akt (S473)65CST 9271 CamKII (T286)52, 60 Promega V1111 p-Erk (T202/Y204)46, 44CST 9101 p-p38MAPK (T180/Y182)42CST 9216 1. Phospho-protein responses in C5a, UDP or Fc  receptor activated RAW 246.7 cells using FXM phospho-specific antibody mix 1. Un, untreated. Phosphorylation state changes are evident for Akt in C5a treated cells; and for p90RSK, Erk1/2 and p38MAPK in UDP treated cells. Anti-Pyk2 (Y402) antibody was not used in this experiment. For Fc  receptor cross-linking, cells were treated with 20  g/ml IgG2a and 66  g/ml F(ab)’ 2. Antibody target MW ( kDa)SourceScore Antibody target MW kDaSourceScore Akt (S473)65CST 92711 p38 MAPK (T180/Y182)42CST 92161 Akt (T308)60CST 92754 p40Phox (T154)40CST 43113 Btk (Y223)77CST 35313 p70 S6 Kinase (T389)68CST 92053 CamKII (T286) 52, 60 Promega V11112/3 p70 S6 Kinase (T421/S424)68CST 92042 c-Cbl (Y731)120CST 35544 Pak1(S199/20 4)/Pak2(S192/ 197) 61-7 /68- 74 CST 26053 cPLA2 (S505)110CST 28313/4 pan-Src (Y416)60CST 21014 creb (S133)43CST 91912/3paxillin (Y118)68 BioSource 44-7223 FAK (Y397)125BD 6118063PDK1 (S241) 61, 69CST 30612/3 FAK (Y576)125BS 44-6523 PDK1/2 (T373/376) 60, 68CST 30653 FAK (Y925)125CST32842 PKCzeta/lamb da (T410/403)76CST 93784 FKHR (T24) /FKHRL1 (T32) 68, 97CST 94642 PLC  3 (S1105)150CST 24844 Gab1 (Y307)115CST 32342 PLC  3 (S537) 150CST 24814 Gab1 (Y627)110CST 32314 PLC  1 (Y783) 155CST 28214 Gab2 (Y452)98CST 38814 PLC  2 (Y1217) 150CST 38712/3 GSK3  /  (S21/9) 46, 52CST 93313Pyk2 (Y402)116CST 32912 Lyn (Y507) 53, 56CST 27313Pyk2 (Y881)132 BioSource 44-6203 MARCKS (S152/156) 39, 68-90Chemicon2Shp-2 (Y542)72CST 37513 mTOR (S2448)289CST 29713Shp-2 (Y580)72CST 37543 mTOR (s2481)289CST 29743Ship-1 (Y1020)145CST 39413 Myosin light chain (S20)19 Rockland 600-401- 4162/3Syk (Y323)70CST 27154 VASP52CST 31113 Syk (Y525/526)70CST 27114 Phospho-antibody survey Scores: 1=unambiguous phospho-response to FXM ligands, little background (in red) 2=weak phospho-response to FXM ligands, extra bands (in green) 3=no phospho-response to FXM ligands, prominent band at expected size 4=no phospho-response to FXM ligands, no prominent band at expected size 2. Changes in protein phosphorylation states in the G  i2 (top and middle panels) or G  q (bottom panel) gene knock-down cells. Immunoreactivity of phospho-Akt, - p38MAPK or - pyk2 were detected with FXM phospho- specific antibody mix 1. The normalized fold changes of ligand-induced responses over responses in untreated samples were measured at each time point. Average responses from three western blots (using the same set of samples) were shown in the top and bottom panels. For pertussis toxin (PTx) treatment, cells were treated with 50 ng/ml PTx at 37 degree for overnight before ligand treatment. These results were also replicated with samples prepared in the Dallas Cell lab. 2. Assessing changes of protein phosphorylations in gene knock-down cells Enhanced p-Akt response to C5a in G  i2 knock-down cells Reduced P-p38MAPK, P-pyk2 responses to UDP in G  q KD cells The enhanced P-Akt response to C5a is PTx sensitive We have evaluated over 40 phospho-specific antibodies for their responses to C5a, UDP and Fcg ligation. Of these, 13 phospho-specific antibodies were selected to form three tentative FXM phospho-specific antibody mixes. Only the antibodies for phospho-Akt and -p38 MAPK detected robust and consistent responses in cells treated with a single ligand. We have examined changes of FXM-ligand induced protein phosphorylation subsequent to gene perturbation. In G  q gene knock-down cells, there were diminished phospho- pyk2 and –p38MAPK responses to UDP, supporting an essential role of G  q in mediating these responses. In contrast, in G  i2 gene knock-down cells, an unexpected increase in C5a induced Akt phosphorylation was observed. Similarly, the C5a –induced calcium responses were also increased in these cells. These data suggest that as yet undetermined compensatory changes are a main form of responses to G  i2 knockdown in these cells. Audra Wendt, Jason Polasek, Katherine Hawes, Richard Davis, Melissa Kachura, David Quan and Carrie Wong for technical assistance; the antibody lab for excellent support and intellectual input; Dr. Paul Sternweis and the macrophage committee for guidance.