SahinRNAi2008              package:nem              R Documentation

_C_o_m_b_i_n_a_t_o_r_i_a_l _P_r_o_t_e_i_n _K_n_o_c_k_d_o_w_n_s _i_n _t_h_e _E_R_B_B _S_i_g_n_a_l_i_n_g _P_a_t_h_w_a_y

_D_e_s_c_r_i_p_t_i_o_n:

     Sixteen RNAi knockdowns (including 3 double knockdowns) of
     proteins in the ERBB signaling pathway of trastuzumab resistant
     breast cancer cells were conducted. Reverse Phase Protein Array
     (RPPA) measurments for 10 signaling intermediates are available
     before and after EGF stimulation with 4 technical and 3 biological
     replicates.

_U_s_a_g_e:

     data(SahinRNAi2008)

_F_o_r_m_a_t:

     dat.unnormalized: 408 x 17 matrix (rows = RPPA measurements for
     (16 KOs + MOCK) x 4 technical x 3 biological replicates, columns =
     10 antibodies + 6 proteins without measurements + time)
     dat.normalized: 408 x 17 matrix (measurements from technical and
     biological replicates are quantile normalized for each RNAi
     experiment)

_D_e_t_a_i_l_s:

     The cells were lysed on ice by scraping the cells in M-PER lysis
     buffer (Pierce, Rockford, IL) containing protease inhibitor
     Complete Mini (Roche, Basel), anti-phosphatase PhosSTOP (Roche,
     Basel), 10 mM NaF and 1mM Na4VO3. Protein concentrations were
     determined with a BCA Protein Assay Reagent Kit (Pierce, Rockford,
     IL). Lysates were mixed 1:2 with 2 times Protein Arraying Buffer
     (Whatman, Brentfort, UK) to obtain a final protein concentration
     of 1.5 mug/muL. Briefly, these lysates were printed onto
     nitrocellulose coated ONCYTE-slides (Grace Bio Labs, Bend, USA)
     using a non-contact piezo spotter, sciFlexxarrayer S5 (Scienion,
     Berlin, Germany). After primary and near-infrared (NIR)-dye
     labeled secondary antibodies applied, spots were analysed using an
     Odyssey scanner (LI-COR, Lincoln, USA) and signal intensities were
     quantified using Odyssey 2.0 software (For detailed information
     and an antibody list, see Sahin et al., 2008). Since no antibody
     against MEK1 was available, we measured protein expression of
     pERK1/2, which is downstream of MEK1.

_R_e_f_e_r_e_n_c_e_s:

     Oezguer Sahin, Holger Froehlich, Christian Loebke, Ulrike Korf,
     Sara Burmester, Meher Majety, Jens Mattern, Ingo Schupp, Claudine
     Chaouiya, Denis Thieffry, Annemarie Poustka, Stefan Wiemann, Tim
     Beissbarth, Dorit Arlt, Modeling ERBB receptor-regulated G1/S
     transition to find novel targets for de novo trastuzumab
     resistance, BMC Systems Biology, 2008

_S_e_e _A_l_s_o:

     'BoutrosRNAi2002'

_E_x_a_m_p_l_e_s:

         data("SahinRNAi2008")
         dim(dat.normalized)
         dim(dat.unnormalized)      

