Journal: BMC Molecular Biology

Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

doi: 10.1186/1471-2199-13-7

Figure Lengend Snippet: Down-regulation of CK2 results in persistent γ-H2AX signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.

Article Snippet: M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described [ ].

Techniques: Transfection, Incubation, Labeling, Staining, Software, Standard Deviation

Journal: BMC Molecular Biology

Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

doi: 10.1186/1471-2199-13-7

Figure Lengend Snippet: CK2 co-localizes with γ-H2AX to sites of DNA damage . A . Association between CK2α' and γ-H2AX was revealed by in situ PLA. Cells expressing or lacking CK2α' were left untreated or incubated with 0.5 μg/ml NCS for 1 hour before fixation and labeling with primary antibodies directed against the indicated proteins. Control, refers to cells transfected with si-CK2α' and stained with secondary antibodies. Cell nuclei were revealed by Hoechst staining. B . Quantification of the number of signals/cell as distinct fluorescent red spots was performed by computer-assisted image analysis as described in Experimental Procedures. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference.

Article Snippet: M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described [ ].

Techniques: In Situ, Expressing, Incubation, Labeling, Transfection, Staining

Journal: BMC Molecular Biology

Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

doi: 10.1186/1471-2199-13-7

Figure Lengend Snippet: Cells overexpressing CK2 exhibit a prompt response to induction of DNA double-strand break . A . M059K cells transiently overexpressing CK2α'-DsRed or DsRed-fluorescent protein were incubated with 0.5 μg/ml NCS for the indicated times. Subsequently, cells were fixed and labeled with anti-γ-H2AX antibody. Cell nuclei were revealed by DAPI staining. Arrows: cells overexpressing CK2α'-DsRed are more efficient in DNA damage repair as compared to cells that do not overexpress the kinase. One representative experiment is shown. B . Average γ-H2AX foci number per red fluorescent cell as described in A . Mean values +/- SD from three independent experiments are shown. More than 100 cells have been analyzed per experiment. *P < 0.001 denotes statistically significant difference. NS, not significant.

Article Snippet: M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described [ ].

Techniques: Incubation, Labeling, Staining

Journal: Cancer Cell International

Article Title: Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells

doi: 10.1186/1475-2867-6-13

Figure Lengend Snippet: Cell cycle and DNA damage response analysis of RC-L1-expressing cells . MCF-7 cells either Mock transfected, expressing GFP or RC-L1 were analyzed by FACS as described under "Experimental Procedures". The histograms represent the distribution of cells through the cell cycle measured by flow cytometry and analyzed with ModFit. (A) untransfected MCF-7 cells. (B) MCF-7 cells transfected with EGFP (C) MCF-7 cells transfected with RC-L1. The percentage of cells in G 2 or M is shown for each treatment group. Detection of the induction of γ-H2AX foci formation using anti-γ-H2AX polyclonal antibody. (D) by immunostaining of Mock transfected cells and serially passaged RC-L1-expressing cells (P1-P4) (E) Number of γ-H2AX foci in four different cell passages, P1-P4. Error bars show s.d. (F). Expression level of γ-H2AX determined by immunoblotting.

Article Snippet: Anti-γ-H2AX rabbit polyclonal antibody from Cell Signaling Technologies and FITC-conjugated secondary antibody from ICN Biomedicals, Inc.

Techniques: Expressing, Transfection, Flow Cytometry, Immunostaining, Western Blot

Journal: Cancer Cell International

Article Title: Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells

doi: 10.1186/1475-2867-6-13

Figure Lengend Snippet: Impact of RC-L1 expression and retrotransposition on p53 mutant cells . (A) Confirmation of L1 retrotransposition by PCR as revealed by a 342 bp product. (B) The histograms represent the distribution of cells through the cell cycle measured by flow cytometry and analyzed with ModFit. "A" untransfected T47D cells. "B" T47D cells transfected with EGFP "C" T47D cells transfected with RC-L1. (C) RC-L1- expression determined by immunofluorescence using anti-L1 ORF1 and anti-L1 ORF2 rabbit polyclonal antibodies (upper panels). Detection of the induction of γ-H2AX nuclear foci formation using anti-γ-H2AX polyclonal antibody (lower panels). (D) Pro-apoptotic bax gene expression level determined by immunoblotting using anti-Bax polyclonal antibody in T47D cells either expressing RC-L1, or treated with 5Gy radiation or both and assessed at the indicated time points.

Article Snippet: Anti-γ-H2AX rabbit polyclonal antibody from Cell Signaling Technologies and FITC-conjugated secondary antibody from ICN Biomedicals, Inc.

Techniques: Expressing, Mutagenesis, Flow Cytometry, Transfection, Immunofluorescence, Western Blot

Journal: EMBO Reports

Article Title: Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA damage repair

doi: 10.15252/embr.202154136

Figure Lengend Snippet:

Article Snippet: Rabbit anti‐H2AX‐P (polyclonal), IF (1:500), WB (1:1,000) , Cell Signaling , Cat #9718; RRID:AB_2118009.

Techniques: Stable Transfection, Transfection, Recombinant, Magnetic Beads, Software, Cell Viability Assay, In Situ

Journal: bioRxiv

Article Title: DNA-PKcs regulates myogenesis in an AKT-dependent manner independent of induced DNA damage

doi: 10.1101/2022.06.23.497315

Figure Lengend Snippet: Quantitative PCR (histogram) analyses of several DNA fragments in the Myogenin promoter from ChIP assays with either (a) anti-MyoD or (b) anti-DNA-PKcs antibodies in differentiating cells Significance by Unpaired t-test (F=2.9, DFn=2, DFd=2). (c) Scheme not at scale of primer positions on the the Myog promoter wih the respective distances from the initiation of transcription site (+1), and summary of positive amplifications in light blue and statistically significant in dark blue (MyoD) or orange (DNA-PKcs) whereas crossed boxes in pink indicate no amplification, in the absence and in the presence of irradition: n=3 unless diffferently indicated. Immunostaining with (d) MyoD, (e) p300, and (f) DNA-PKcs of immunoprecipitation with DNA-PKcs and Akt1 (IgG rabbit, IgR), and Akt2 (IgG Mouse, IgM) pull-down. Input, empty IgG rabbit (IgGR, for control of DNA-PKcs and Akt1) and IgGM (IgG Mouse, IgM, for control of Akt2) in C2C7 cells at 5dps. g) Schematic representation of the interactions (dotted lines) observed in immuniprecipiatation experiments; dotted lines of the same colour indicate interactions detected with pulldown of the same protein. Protein sizes are not at scale. Histone ψH2AX immunostaining evaluated per cell in the absence [control, vehicle (0.2% DMSO)] and in the presence of 10 µM DNAPKi at different time points h) in non-irradiated cells and i ) irradiated C2C7 cells. Total ψH2AX immunofluorescence intensity was assessed with ImageJ of single cells after acquisition with Cell Voyager CV1000, confocal scanner box (Yokogawa). N= 40-80 cells from 3 independent experiments. Significance by 1-way ANOVA (Non-irradiated: F=16.37, DFn=11, DFd=588, p<0.0001. Irradiated: F=53.68, DFn=11, DFd=457, p<0.0001), with post-hoc Tukey’s multiple comparisons test. P-values are indicated on the histogram. j) WB of Myogenin, global histone H2AX, ψH2AX, and the housekeeping GAPDH protein in C2C7 cells at 4.5 dps. The ratio ψH2AX/H2AX is shown in the bottow line after superimposition of the (700 nm (Goat α-mouse Starbright blue 700) and 800nm (goat α-rabbit CF770) signal with the imagelab program of ChemiDoc MP imaging system (Biorad). The highest doses of DNAPKi results in slightly reduced levels of ψ-H2AX, compatibly with H2AX being also phosphorylated by DNA-PKcs. k ) Enumeration of Myogenin + and Myogenin - cells upon immunostaining per image in the presence and in the absence of inhibitors of DNA-PKcs and caspases (4 images/condition analysed, n=1). Uncropped gels are shown in Fig S5.

Article Snippet: The following antibodies have been used in this study: mouse monoclonal α-Pax7 (AB_528428), mouse monoclonal α−Myogenin (F5D, AB_2146602), mouse monoclonal α-Myosin Heavy chain, MHC (MF20, AB_2147781) and mouse monoclonal α-embryonic MyHC (F1.652, AB_528358) antibodies from Developmental Studies Hybridoma Bank; rabit polyclonal α-Myogenin (sc-576), rabbit polyclonal α-MyoD (sc-304), Rabbit polyclonal α-GAPDH (sc-25778) from Santa Cruz; mouse monoclonal α-Akt2 (5239), rabbit polyclonal α-phospho-Akt2 (Ser474) (8599), rabbit polyclonal α-Akt1 (C73H10), rabbit polyclonal α-phospho-Akt1 (Ser473) (9018), rabbit polyclonal α-phospho-Akt (9271), rabbit polyclonal α-H2AX (2595) from Cell Signaling technology; mouse monoclonal α-phospho-H2AX(Ser139) (05-636) from Millipore; rabbit polyclonal α-53BP1 (NB100-304) from Novusbio; chicken polyclonal-α-GFP (ab13970), rabbit polyclonal-α-DNAPKcs (ab70250) rabbit monoclonal α-Cyclin A2 (ab181591) from abcam; Rabbit α-Laminin (L9393), Propodium Iodide (P4864) from Sigma; goat α-rabbit fluor 555 (A21428), goat α-rabbit fluor 488 (A11034), goat α-mouse fluor 488 (A11029), goat α-mouse fluor 555 (A21425), goat α-mouse fluor cy5 (A10524), goat α-chicken fluor 488 (A11039), goat α-mouse-HRP (31430), goat α-rabbit-HRP (31460) from Invitrogen; hFAB Rhodamine Anti-GAPDH IgG (12004168), Goat α-mouse Starbright blue 700 (12004159) from Biorad; goat α-rabbit CF770 (10078-1) from Biotium; FITC Annexin V Apoptosis Detection kit I (556547) from BD Biosciences.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Immunostaining, Immunoprecipitation, Irradiation, Immunofluorescence, Imaging

Journal: JCI Insight

Article Title: DNA damage is overcome by TRIP13 overexpression during cisplatin nephrotoxicity

doi: 10.1172/jci.insight.139092

Figure Lengend Snippet: Following cisplatin treatment (15 mg/kg IP), kidneys were harvested after 1 or 3 days for immunohistochemistry of ( A – H ) γ-H2AX (Ser139) and ( I – L ) 8-hydroxy-2′-deoxyguanosine (8-OHdG). After 24 ( A and B ) and 72 ( C – F ) hours following cisplatin administration, γ-H2AX (Ser139) was detected in ( A – D ) Trip13 Stop/Stop and ( E and F ) Trip13 ΔStop mice and quantified by counting positive nuclei. Sections were counterstained with hematoxylin. Negative control (no primary antibody) is shown ( G ) using sections from Trip13 ΔStop mice treated with cisplatin at day 3. ( H ) Graphical analysis of γ-H2AX (Ser139) as a percentage of total nuclei. — = vehicle; + = cisplatin. *** P < 0.001, **** P < 0.0001 between the indicated groups using 1-way ANOVA with Tukey’s post hoc analysis. ( I – L ) Immunofluorescence of 8-OHdG in kidney sections from vehicle- and cisplatin-treated Trip13 Stop/Stop and Trip13 ΔStop mice after day 3. Alexa Fluor 555 (red) fluorescence was used to detect 8-OHdG, and Alexa Fluor 488 (green) fluorescence was used to detect proximal tubule lectin (PVA-E). DAPI was used to detect nuclei (blue). Scale bar: 200 μm ( A – G ); 100 μm ( I – L ). n = 4–8 animals per group.

Article Snippet: The following primary monoclonal mouse antibody against PCNA (catalog 2586; 1:250; Cell Signaling Technology), and primary polyclonal rabbit antibodies against phospho-H2AX (Ser139) (catalog 9718; 1:250, Cell Signaling Technology), phospho–histone H3 (Ser10; catalog 9701; 1:250; Cell Signaling Technology), cleaved caspase-3 (catalog 9661, 1:200; Cell Signaling Technology), and Ki67 (catalog 15580, 1:200; Abcam) were used on the kidney sections.

Techniques: Immunohistochemistry, Negative Control, Immunofluorescence, Fluorescence

Journal: JCI Insight

Article Title: DNA damage is overcome by TRIP13 overexpression during cisplatin nephrotoxicity

doi: 10.1172/jci.insight.139092

Figure Lengend Snippet: ( A and B ) Nanoparticles (50 μg) containing fluorescent ICG dye were injected into mice and monitored 24 hours later using IVIS system in ( A ) living mice and ( B ) ex vivo excised organs. ( C ) Ex vivo organ fluorescence accumulation ( n = 3 animals). K, kidney; Lu, lung; Liv, liver; H, heart; Spl, spleen. Data represent mean ± SD. ( D – G ) Representative histological images from ( D ) vehicle- or ( E and F ) cisplatin-treated (15 mg/kg IP) Trip13 Stop/Stop ( D and E ) and Trip13Δ Stop ( F ) mice. ( G ) Tubular epithelial cell damage was scored as a percentage of total tubules counted. Serum markers of AKI were monitored for ( H ) creatinine and ( I ) NGAL after 72 hours following treatment with cisplatin (15 mg/kg IP) and NP-mirin or NP-Ctrl (50 mg/kg IP). ( J ) γ-H2AX– (Ser139) and ( K ) PCNA-positive nuclei in kidney sections at 72 hours following cisplatin treatment with and without mirin. NP without mirin (50 mg/kg IP) was used as the control solution. ( L ) Relative gene expression change following treatment with cisplatin and/or mirin. Each of the respective groups was compared with mouse kidney values obtained from control vehicle-treated Trip13 Stop/Stop mice. ( G – K ) n = 5–7 mice/group; ( L ) n =4 mice/group. * P < 0.05, ** P < 0.01 between all organs ( C ) or indicated groups ( G , I , J , and L ) or all groups ( C ) using 1-way ANOVA with Tukey’s post hoc analysis. Significance for creatinine ( H ) was determined using Kruskal-Wallis nonparametric test with Dunn’s post hoc analysis. Scale bar: 100 μm. + = administration of cisplatin or mirin; Cre - = Trip13 Stop/Stop ; Cre + = Δ Stop.

Article Snippet: The following primary monoclonal mouse antibody against PCNA (catalog 2586; 1:250; Cell Signaling Technology), and primary polyclonal rabbit antibodies against phospho-H2AX (Ser139) (catalog 9718; 1:250, Cell Signaling Technology), phospho–histone H3 (Ser10; catalog 9701; 1:250; Cell Signaling Technology), cleaved caspase-3 (catalog 9661, 1:200; Cell Signaling Technology), and Ki67 (catalog 15580, 1:200; Abcam) were used on the kidney sections.

Techniques: Injection, Ex Vivo, Fluorescence, Expressing

Journal: JCI Insight

Article Title: DNA damage is overcome by TRIP13 overexpression during cisplatin nephrotoxicity

doi: 10.1172/jci.insight.139092

Figure Lengend Snippet: NU7441 (20 mg/kg) was administered IP to Trip13 Stop/Stop and Trip13Δ Stop mice that were euthanized prior to or at 48 hours to collect blood and kidneys. ( A – D ) Representative histological images from ( A ) vehicle- or ( B – D ) cisplatin-treated (15 mg/kg IP) Trip13 Stop/Stop ( A – C ) and Trip13Δ Stop ( D ) mice coadministered with either vehicle ( B ) or NU7441 (20 mg/kg IP; A , C , and D ). ( E ) Tubular epithelial cell damage was scored as a percentage of total tubules. ** P < 0.01 between all groups. ( F ) Serum markers of AKI were measured for creatinine after 48 hours following treatment with cisplatin (15 mg/kg IP). * P < 0.05; ** P < 0.01 between cisplatin- and NU7441-treated mice. Positive nuclei for ( G ) γ-H2AX (Ser139) and ( H ) Ki-67 in kidney sections at 48 hours following cisplatin treatment with and without NU7441. * P < 0.05 between both genetic strains treated with cisplatin- and NU7441; *** P < 0.001 between all groups. One-way ANOVA with Tukey’s post hoc analysis was performed for each set of data ( E – H ). n = 4–7 mice/group. Scale bar: 100 μm. Δ Stop, Trip13Δ Stop mice; Trip13 St/St , Trip13 Stop/Stop mice.

Article Snippet: The following primary monoclonal mouse antibody against PCNA (catalog 2586; 1:250; Cell Signaling Technology), and primary polyclonal rabbit antibodies against phospho-H2AX (Ser139) (catalog 9718; 1:250, Cell Signaling Technology), phospho–histone H3 (Ser10; catalog 9701; 1:250; Cell Signaling Technology), cleaved caspase-3 (catalog 9661, 1:200; Cell Signaling Technology), and Ki67 (catalog 15580, 1:200; Abcam) were used on the kidney sections.

Techniques:

Journal: Oncology Letters

Article Title: Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

doi: 10.3892/ol.2021.12740

Figure Lengend Snippet: DNA damage response signaling is impaired in Jurkat cells, but the induction of apoptosis is retained. Jurkat cells treated with DMSO, 1 µg/ml Doxo or 5 µg/ml Eto for 24 h or 72 h were analyzed for (A) apoptosis using Annexin V/7AAD and (B) caspase-3/7 activity using a Muse Cell Analyzer, and (C) subjected to the western blot analysis of Wip1, p-ATM, T-ATM, ATR, p-Chk1, T-Chk1, p-Chk2, T-Chk2, gH2AX and H2AX. GAPDH was used as the loading control. Data shown are the means ± SD of three independent experiments. The statistical significance of differences in the data was analyzed using one-way ANOVA followed with Bonferroni multiple comparison tests. *P≤0.001 vs. C; # P≤0.001 vs. Eto. C, DMSO control; Doxo, doxorubicin; Eto, etoposide; Wip1, p53-induced phosphatase 1; p-, phospho-; T-, total; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiestasia and Rad3-related; Chk, checkpoint kinase; H2AX, H2A histone family member X; gH2AX, p-H2AX (S139).

Article Snippet: Mouse anti-Wip1 (F-10; cat. no. sc-376257; 1:250), mouse anti-Chk1 (G-4; cat. no. sc-8408; 1:250), mouse anti-Chk2 primary antibodies (cat. no. sc-17747; 1:250) and horseradish peroxidase-coupled anti-mouse (cat. no. sc-2357; 1:1,000) or anti-rabbit (cat. no. sc-2004; 1:1,000) secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Polyclonal rabbit anti-phosphorylated (p)-Chk1 (S345; cat. no. 2348), polyclonal rabbit anti-p-Chk2 (T68; cat. no. 2197), monoclonal mouse anti-ATM (cat. no. 2873), rabbit anti-p-ATM (S1981; cat. no. 5883), monoclonal mouse anti-ATR (cat. no. 2790), polyclonal rabbit anti-p-ATR (S428; cat. no. 2853) antibody and polyclonal rabbit anti-H2AX (cat. no. 7631) and anti-γH2AX (S139; cat. no. 9718) were purchased from Cell Signaling Technology, Inc. (all 1:1,000).

Techniques: Activity Assay, Western Blot

Journal: Oncology Letters

Article Title: Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

doi: 10.3892/ol.2021.12740

Figure Lengend Snippet: Induction of senescence is unaffected by increased Wip1 in Jurkat cells. Jurkat cells were treated with DMSO, 0.2 µg/ml Doxo or 1 µg/ml Eto for 72 h and the induction of senescence was assayed by measuring (A) cell viability by WST-1 assay, (B) the cell cycle profile and (C) the amount of active H2AX [γH2AX; p-H2AX (S139)] and non-active (unphosphorylated) H2AX. (D) Staining of cells for senescence-associated β-galactosidase activity. Scale bar, 50 mm. (E) Western blot analysis of Wip1, p-ATM, T-ATM, ATR, p-Chk1, T-Chk1, p-Chk2 and T-Chk2. GAPDH was used as the loading control. Data shown are the means ± SD of three independent experiments. The statistical significance of differences in the data was analyzed by one-way ANOVA followed by Bonferroni multiple comparison tests. *P≤0.001 vs. C. C, DMSO control; Doxo, doxorubicin; Eto, etoposide; H2AX, H2A histone family member X; Wip1, p53-induced phosphatase 1; p-, phospho-; T-, total; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiestasia and Rad3-related; Chk, checkpoint kinase.

Article Snippet: Mouse anti-Wip1 (F-10; cat. no. sc-376257; 1:250), mouse anti-Chk1 (G-4; cat. no. sc-8408; 1:250), mouse anti-Chk2 primary antibodies (cat. no. sc-17747; 1:250) and horseradish peroxidase-coupled anti-mouse (cat. no. sc-2357; 1:1,000) or anti-rabbit (cat. no. sc-2004; 1:1,000) secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Polyclonal rabbit anti-phosphorylated (p)-Chk1 (S345; cat. no. 2348), polyclonal rabbit anti-p-Chk2 (T68; cat. no. 2197), monoclonal mouse anti-ATM (cat. no. 2873), rabbit anti-p-ATM (S1981; cat. no. 5883), monoclonal mouse anti-ATR (cat. no. 2790), polyclonal rabbit anti-p-ATR (S428; cat. no. 2853) antibody and polyclonal rabbit anti-H2AX (cat. no. 7631) and anti-γH2AX (S139; cat. no. 9718) were purchased from Cell Signaling Technology, Inc. (all 1:1,000).

Techniques: WST-1 Assay, Staining, Activity Assay, Western Blot