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)      

