Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Protein Targeting of Phospholamban: A Common Role for an N-Terminal Di-Arginine Motif in ER Retention?

doi: 10.1371/journal.pone.0011496

Figure Lengend Snippet: ( A ) Subcellular fractionation of wild type Sigma 1R and a di-arg to di-glu mutant Sigma 1R construct. HEK cells were transiently transfected with tagged V5 Sigma 1R or tagged V5 di-arg to di-glu mutant Sigma 1R and cell lysates run on a continuous 20–60% sucrose gradient. Sigma1R was detected using the anti-V5 antibody. ( B ) IP performed using cell lysates from wild type V5 tagged Sigma 1R and V5 tagged di-arginine mutant Sigma 1R constructs transfected HEK cells. Proteins were immunoprecipitated using anti COP I antibody and then immunoblotted with anti V5 antibody (right panel); with equivalent loading of lysates (left panel). Arrows indicate Sigma 1R and mutant Sigma 1R. ( C ) Subcellular fractionation of wild type SR-β and di-arginine mutant SR-β construct. HEK cells were transiently transfected with V5 tagged SR-β or V5 tagged di-arginine mutant SR-β and cell lysates run on a continuous 20–60% sucrose gradient. SRB was detected using anti-V5 antibody. ( D ) IP performed using cell lysates from wild type V5 tagged SR-β and V5 tagged di-arginine mutant SR-β constructs transfected HEK cells and immunoprecipitated using anti COP I antibody and then immunoblotted with anti V5 antibody (right panel); with equivalent loading of lysates (left panel). Arrows indicate SR-β and mutant SR-β ( E ) Subcellular fractionation of wild type SOAT and di-arginine mutant SOAT construct. HEK cells were transiently transfected with tagged SOAT and di-arginine mutant SOAT and cell lysates run on a continuous 20–60% sucrose gradient and detected using anti-SOAT antibody ( F ) Tandem affinity purification of HEK cells transiently transfected with tagged SOAT and di-arginine mutant SOAT. Purification was carried out using streptavidin beads first and calmodulin beads second. Samples were then probed with antibodies to detect COP I, arrows indicate COP I (see for details).

Article Snippet: Due to cloning difficulties, SOAT cDNA (OriGene) was cloned into the Interplay Mammalian TAP vector, pCTAP (Stratagene) which contains a dual streptavidin binding peptide tag and a calmodulin binding peptide tag.

Techniques: Fractionation, Mutagenesis, Construct, Transfection, Immunoprecipitation, Affinity Purification, Purification

Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: APC11 depletion promotes CUL5 neddylation but stabilizes integrin β1. a) Immunoblot of indicated proteins in PLC/PRF/5, Hep3B, and PANC‐1 cells transfected with siRNA targeting APC11 or with a scrambled control siRNA. b) Co‐IP of transfected FLAG‐CUL5 and endogenous CAND1, COPS5, UBE2F or SAG in HEK293 cells transfected with indicated siRNA oligos. c) Co‐IP of endogenous CUL5 with UBE2F or APC11 in A549 cells transfected with indicated siRNA oligos. d) Immunoblot of CUL5 in A549 and PLC/PRF/5 cells transfected with indicated siRNA oligos. e) The stability of SOCS3 and integrin β1 in A549 and MIA PaCa‐2 cells upon APC11 knockdown. A549 and MIA PaCa‐2 cells were transfected with indicated siRNA oligos for 72 h and then treated with 100 µg mL −1 CHX for the indicated time periods, followed by IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right). Data are presented as mean ± SEM, n = 3. For statistical analysis, significances were determined by Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. f) Diagram illustrating how APC11 depletion promotes CUL5 neddylation and leads to integrin β1 accumulation.

Article Snippet: [ ] The following antibodies were used: ACTIN (Sigma–Aldrich, A5441), FLAG (Sigma–Aldrich, F1804), FLAG (Sigma–Aldrich, F7425), GFP (Santa Cruz, sc‐9996), MYC (Santa Cruz, sc‐789), APC2 (Cell Signaling Technology, 12301), APC8 (Cell Signaling Technology, 15100), APC10 (Cell Signaling Technology, 14807), APC11 (Cell Signaling Technology, 14090), APC11 (Santa Cruz, sc‐517142), SAG (Proteintech, 11905‐1‐AP), RBX1 (Proteintech, 14895‐1‐AP), CUL1 (Santa Cruz, sc‐11384), CUL5 (Abcam, ab184177), CUL5 (Santa Cruz, sc‐373822), CAND1 (Abcam, ab183748), integrin β1 (Cell Signaling Technology, 34 971), p‐SRC (Y416) (Cell Signaling Technology, 6943), ATM (Cell Signaling Technology, 2873), Cyclin B1 (Cell Signaling Technology, 4138), RPB1 (Cell Signaling Technology, 14958), EGFR (Cell Signaling Technology, 4267), DEPTOR (Cell Signaling Technology, 11 816), NOXA (Millipore, OP180), UBE2F (Proteintech, 17056‐1‐AP), COPS5 (Cell Signaling Technology, 6895), SOCS3 (Proteintech, 14025‐1‐AP), LC3 (Cell Signaling Technology, 2775), securin (Cell Signaling Technology, 13445), ITCH (Proteintech, 20920‐1‐AP), Cyclin E1 (Cell Signaling Technology, 20808), p‐H3 (Cell Signaling Technology, 3377), and p‐Aurora A/B/C (Cell Signaling Technology, 2914), Aurora A (Cell Signaling Technology, 14 475), Aurora B (Abcam, ab2254), and NEDD8 (Abcam, ab81264).

Techniques: Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Knockdown

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: Neuroreport

Article Title: Cathepsin B promotes optic nerve axonal regeneration

doi: 10.1097/WNR.0000000000002148

Figure Lengend Snippet: Western blot analysis of microglial protein expression following IL-4 and IL-6 activation. (a) Grayscale images depicting the expression levels of STAT3, STAT6, IRE1α, and CTSB in microglia following activation induced by IL-4 and IL-6, as assessed by western blot analysis. (b) The statistical analysis results indicate that, in comparison with the control group, the expression of STAT3, STAT6, IRE1α, and CTSB proteins in microglia is significantly upregulated in the IL-4 + IL-6 group. N = 3, P < 0.05. CTSB, cathepsin B; IL, interleukin; IRE1α, inositol-requiring enzyme 1 alpha; STAT3, signal transducer and activator of transcription 3; STAT6, signal transducer and activator of transcription 6.

Article Snippet: The primar antibodies utilized were: anti-fibronectin (15613-1-AP; Proteintech), antineurocan (1 : 1000; 27087-1-AP, Proteintech), anti-STAT3 (1 : 1000; 10253-2-AP, Proteintech), anti-STAT6 (1 : 1000; 82630-1-RR, Proteintech), anti-IRE1α (1 : 1000; 27528-1-AP, Proteintech), and anti-CTSB (1 : 1000; 12216-1-AP, Proteintech).

Techniques: Western Blot, Expressing, Activation Assay, Comparison, Control

Journal: Neuroreport

Article Title: Cathepsin B promotes optic nerve axonal regeneration

doi: 10.1097/WNR.0000000000002148

Figure Lengend Snippet: Immunofluorescence analysis of microglial protein expression following IL-4 and IL-6 activation. (a) The expression levels of STAT3, STAT6, IRE1α, and CTSB in microglia following activation induced by IL-4 and IL-6 were assessed using immunofluorescence methods. (b) The statistical analysis results indicate that, in comparison with the control group, the expression of STAT3, STAT6, IRE1α, and CTSB proteins in microglia is significantly upregulated in the IL-4 + IL-6 group. N = 3, P < 0.05, scale bar = 50 μm. CTSB, cathepsin B; IL, interleukin; IRE1α, inositol-requiring enzyme 1 alpha; STAT3, signal transducer and activator of transcription 3; STAT6, signal transducer and activator of transcription 6.

Article Snippet: The primar antibodies utilized were: anti-fibronectin (15613-1-AP; Proteintech), antineurocan (1 : 1000; 27087-1-AP, Proteintech), anti-STAT3 (1 : 1000; 10253-2-AP, Proteintech), anti-STAT6 (1 : 1000; 82630-1-RR, Proteintech), anti-IRE1α (1 : 1000; 27528-1-AP, Proteintech), and anti-CTSB (1 : 1000; 12216-1-AP, Proteintech).

Techniques: Immunofluorescence, Expressing, Activation Assay, Comparison, Control