1. FXM phospho-specific antibody mix 1 Introduction Summary Gene perturbation by RNAi: See posters from the Molecular Biology lab and the Macrophage Biology.

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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 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 lysates for western blot analysis: PP Protein transfer: PP Western blot analysis for RAW 264.7: PP 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 Phospho-protein responses in C5a, UDP or Fc  receptor activated RAW 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 p38 MAPK (T180/Y182)42CST Akt (T308)60CST p40Phox (T154)40CST Btk (Y223)77CST p70 S6 Kinase (T389)68CST CamKII (T286) 52, 60 Promega V11112/3 p70 S6 Kinase (T421/S424)68CST c-Cbl (Y731)120CST Pak1(S199/20 4)/Pak2(S192/ 197) 61-7 / CST cPLA2 (S505)110CST 28313/4 pan-Src (Y416)60CST creb (S133)43CST 91912/3paxillin (Y118)68 BioSource FAK (Y397)125BD PDK1 (S241) 61, 69CST 30612/3 FAK (Y576)125BS PDK1/2 (T373/376) 60, 68CST FAK (Y925)125CST32842 PKCzeta/lamb da (T410/403)76CST FKHR (T24) /FKHRL1 (T32) 68, 97CST PLC  3 (S1105)150CST Gab1 (Y307)115CST PLC  3 (S537) 150CST Gab1 (Y627)110CST PLC  1 (Y783) 155CST Gab2 (Y452)98CST PLC  2 (Y1217) 150CST 38712/3 GSK3  /  (S21/9) 46, 52CST 93313Pyk2 (Y402)116CST Lyn (Y507) 53, 56CST 27313Pyk2 (Y881)132 BioSource MARCKS (S152/156) 39, 68-90Chemicon2Shp-2 (Y542)72CST mTOR (S2448)289CST 29713Shp-2 (Y580)72CST mTOR (s2481)289CST 29743Ship-1 (Y1020)145CST Myosin light chain (S20)19 Rockland /3Syk (Y323)70CST VASP52CST Syk (Y525/526)70CST 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.