C myc, by Roche - Product details

1

Inhibition of ERK and c-MYC by ASP5878

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were seeded in 100 mm dishes at 2 × 10⁶ cells/10 mL per dish and cultured overnight. Media were replaced with ASP5878 containing media at the final concentrations of 0, 1, 10, 100 and 1000 nmol/L respectively. The final concentration of DMSO in each dish was 0.01%. Following 2‐h (for ERK and phospho‐ERK) or 48‐h (for c‐MYC) incubation with ASP5878, cells were rinsed with PBS and collected. The cells were lysed with cell lysis buffer (Cell Signaling Technology) containing phosphatase inhibitor (Thermo Scientific) and protease inhibitor (Roche), and protein levels of ERK, c‐MYC and actin, and phosphorylation levels of ERK were determined by immunoblotting. Antibodies were obtained from following sources: ERK (#9102; Cell Signaling Technology) and phospho‐ERK (Thr202/Tyr204) (#9101; Cell Signaling Technology), actin (A5441; Sigma‐Aldrich, St Louis, MO, USA), c‐MYC (#5605; Cell Signaling Technology).

Kikuchi A., Suzuki T., Nakazawa T., Iizuka M., Nakayama A., Ozawa T., Kameda M., Shindoh N., Terasaka T., Hirano M, & Kuromitsu S. (2017). ASP5878, a selective FGFR inhibitor, to treat FGFR3‐dependent urothelial cancer with or without chemoresistance. Cancer Science, 108(2), 236-242.

+ Open protocol

+ Expand

2

Immunofluorescence Staining and Imaging Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunofluorescence slides were prepared according to the protocol of Atkin (1998) . Nuclear staining was performed with DAPI or Hoechst 33342. Primary antibodies were c-Myc (monoclonal, from mouse, by Roche) and c-Myc (polyclonal, from rabbit, by Santa Cruz). Secondary antibody was Alexa Fluor 555 (anti rabbit; Life Technologies). Spheroblasting of cells was done with Zymolyase 100T (Seikagaku). Cells expressing GFP-fused proteins were resuspended in PBS containing 4% formaldehyde and dropped on polyline-coated slides. After 5 min, the slides were washed with PBS, and the cells were coated with 10 μl Hoechst 33342 (5 μg/ml) and 10 μl Mounting Medium (according to Atkin [1998] ) and coverslips.

Reinhardt-Tews A., Krutyhołowa R., Günzel C., Roehl C., Glatt S, & Breunig K.D. (2020). A double role of the Gal80 N terminus in activation of transcription by Gal4p. Life Science Alliance, 3(12), e202000665.

+ Open protocol

+ Expand

3

Western Blotting of Protein Samples

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein samples were subjected to SDS-PAGE gels (Bio-Rad), electrophoresed, transferred to Immobilon-P PVDF (Millipore) or Immobilon-FL PVDF membranes (Millipore) and Western blotting (WB). The following primary antibodies were used: PS1-NTF (a kind gift of Merck Research Laboratories); PS1-CTF (MAB5232, Millipore); PS2-CTF (1987–1, Epitomics); nicastrin was generated in our laboratory; Aph-1aL (38–3600, Invitrogen); Pen-2 (ab18189, Abcam); human IFITM3 (anti-Fragilis, ab109429, Abcam); mouse IFITM3 (anti-Fragilis, ab15592, Abcam); APP (22C11, MAB348, Millipore); SPP (317) was generated in our laboratory; cleaved Notch1 (Val1744), Cell Signaling Technology and SM320, generated in our lab); c-myc (9E10, Roche Life Science); N-cadherin (D4R1H, #13116, Cell Signaling Technology); Rab7 (B-3, sc-376362, Santa Cruz Biotechnology); EEA1 (Ab2900, Abcam); β-actin (C4, sc-47778, Santa Cruz Biotechnology); β-tubulin III (T8578, Sigma-Aldrich); tubulin (ab56676, Abcam). HRP-conjugated anti-rabbit and mouse secondary antibodies (NA9340V, NXA931V, GE Healthcare) for ECL substrate (Pierce) and IRDye 800CW goat anti-rabbit IgG (H+L) or anti-mouse IgG (H+L) secondary antibodies (925–32211, 925–32210, LI-COR) for Odyssey CLx Imaging (LI-COR) were used. For quantification, ImageJ and Image Studio Lite (LI-COR) were used, respectively (for source gels see Supplementary Figure 1).

Hur J.Y., Frost G.R., Wu X., Crump C., Pan S.J., Wong E., Barros M., Li T., Nie P., Zhai Y., Wang J.C., TCW J., Guo L., McKenzie A., Ming C., Zhou X., Wang M., Sagi Y., Renton A.E., Esposito B.T., Kim Y., Sadleir K.R., Trinh I., Rissman R.A., Vassar R., Zhang B., Johnson D.S., Masliah E., Greengard P., Goate A, & Li Y.M. (2020). Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease. Nature, 586(7831), 735-740.

+ Open protocol

+ Expand

4

Western Blot Protein Extraction and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were harvested, washed with ice-cold PBS, and then lysed with lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.5 mM Na orthovanadate, and 0.5% Triton X-100) to prepare protein extracts. Protein concentrations were measured by the Bradford method using bovine serum albumin as standard [29] (link). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane and were probed with specific primary antibody kept overnight at 4°C with gentle rocking. Antibodies against FBXO31, Flag, β-actin, and α-tubulin were procured from Sigma. TP53 antibody was purchased from Santa Cruz, Slug from Abcam, E-cadherin from BD Biosciences, and c-myc from Roche. Snail, Twist, and K48-ubiquitin antibodies were purchased from Cell Signaling Technology. β-actin and α-tubulin were used as loading control.

Manne R.K., Agrawal Y., Bargale A., Patel A., Paul D., Gupta N.A., Rapole S., Seshadri V., Subramanyam D., Shetty P, & Santra M.K. (2017). A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer. Neoplasia (New York, N.Y.), 19(6), 483-495.

+ Open protocol

+ Expand

5

Analysis of Protein Interactions and Modifications

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein lysates were prepared in RIPA cell lysis buffer (Sigma-Aldrich) containing a protease inhibitor mixture (Roche). Equal amounts of protein samples were subjected to electrophoresis and then transferred to a PVDF membrane. After blocking the membrane for 1 h with a PBS-T buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO4, 1.8 mM KH₂PO4, 0.1% Tween 20) containing 2% skimmed milk and 0.1% horse serum (HS) it was incubated with primary antibodies against c-myc (Clone 9E10, Roche Diagnostics), Nurr1 (prepared in our laboratory)⁵⁷ and β-actin (Abcam) overnight at 4 °C. After incubation with secondary antibody for 1 h, immunoblot detection was achieved using the Novex ECL HRP Chemiluminescent substrate. Band intensities were determined using ImageJ software (v.2.0.0-rc-69/1.52n; NIH) and then normalized against β-actin values. For co-IP, protein samples were prepared from collected cells and each protein sample (40 μg) was incubated with mouse anti-c-myc (1:1,000; Sigma-Aldrich) and GammaBind G Sepharose slurry (Sigma-Aldrich) for IP overnight at 4 °C with gentle rotation. After washing, samples were subjected to immunoblot against Nurr1 (1:1,000).

Rajan S., Jang Y., Kim C.H., Kim W., Toh H.T., Jeon J., Song B., Serra A., Lescar J., Yoo J.Y., Beldar S., Ye H., Kang C., Liu X.W., Feitosa M., Kim Y., Hwang D., Goh G., Lim K.L., Park H.M., Lee C.H., Oh S.F., Petsko G.A., Yoon H.S, & Kim K.S. (2020). PGE1 and PGA1 bind to Nurr1 and activate its transcriptional function. Nature chemical biology, 16(8), 876-886.

+ Open protocol

+ Expand

6

Immunofluorescence Staining of Transfected Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Transfected HEK293 and N2a cells were fixed with 4% PFA in phosphate-buffered saline (PBS) and blocked with Tris-buffered saline (TBS) containing 0.2% Triton, 5% goat serum and 5% horse serum.
All antibody incubations and wash steps were performed with TBS. Primary antibodies were c-myc (mouse IgG1K, 9E10, monoclonal, 1:100, #11 667 149 001, Roche), mCherry (rat IgG2a, 16D7, monoclonal, 1:2000, #M11217, ThermoFisher) and HA high affinity (rat IgG1, 3F10, monoclonal, 1:100, #11 867 431 001, Roche). The following highly cross-purified secondary antibodies were purchased from Jackson ImmunoResearch and used at a concentration of 1:250: DyLight™ 405-conjugated donkey anti rabbit (#711-475-152), Cy™3-conjugated donkey anti mouse (#715-165-151), Cy™3-conjugated donkey anti rat (#712-165-153), Alexa Fluor ® 647-conjugated donkey anti mouse (#715-605-151), Alexa Fluor ® 647-conjugated donkey anti rat (#712-605-153), and Cy™5-conjugated donkey anti rat (#712-175-153) . Nuclei were counterstained with DAPI, and cells were mounted with Mowiol containing 2.5% DABCO (1,4-diazabicyclo[2.2.2]octane).

Probst S., Riese F., Kägi L., Krüger M., Russi N., Nitsch R.M, & Konietzko U. (2021). Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity. Biological chemistry, 402(4).

+ Open protocol

+ Expand

7

Western Blotting of Protein Samples

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein samples were subjected to SDS-PAGE gels (Bio-Rad), electrophoresed, transferred to Immobilon-P PVDF (Millipore) or Immobilon-FL PVDF membranes (Millipore) and Western blotting (WB). The following primary antibodies were used: PS1-NTF (a kind gift of Merck Research Laboratories); PS1-CTF (MAB5232, Millipore); PS2-CTF (1987–1, Epitomics); nicastrin was generated in our laboratory; Aph-1aL (38–3600, Invitrogen); Pen-2 (ab18189, Abcam); human IFITM3 (anti-Fragilis, ab109429, Abcam); mouse IFITM3 (anti-Fragilis, ab15592, Abcam); APP (22C11, MAB348, Millipore); SPP (317) was generated in our laboratory; cleaved Notch1 (Val1744), Cell Signaling Technology and SM320, generated in our lab); c-myc (9E10, Roche Life Science); N-cadherin (D4R1H, #13116, Cell Signaling Technology); Rab7 (B-3, sc-376362, Santa Cruz Biotechnology); EEA1 (Ab2900, Abcam); β-actin (C4, sc-47778, Santa Cruz Biotechnology); β-tubulin III (T8578, Sigma-Aldrich); tubulin (ab56676, Abcam). HRP-conjugated anti-rabbit and mouse secondary antibodies (NA9340V, NXA931V, GE Healthcare) for ECL substrate (Pierce) and IRDye 800CW goat anti-rabbit IgG (H+L) or anti-mouse IgG (H+L) secondary antibodies (925–32211, 925–32210, LI-COR) for Odyssey CLx Imaging (LI-COR) were used. For quantification, ImageJ and Image Studio Lite (LI-COR) were used, respectively (for source gels see Supplementary Figure 1).

Hur J.Y., Frost G.R., Wu X., Crump C., Pan S.J., Wong E., Barros M., Li T., Nie P., Zhai Y., Wang J.C., TCW J., Guo L., McKenzie A., Ming C., Zhou X., Wang M., Sagi Y., Renton A.E., Esposito B.T., Kim Y., Sadleir K.R., Trinh I., Rissman R.A., Vassar R., Zhang B., Johnson D.S., Masliah E., Greengard P., Goate A, & Li Y.M. (2020). Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease. Nature, 586(7831), 735-740.

+ Open protocol

+ Expand

8

Western Blotting of Signaling Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed on ice with radioimmunoprecipitation assay (RIPA) buffer containing a proteinase inhibitor (Roche) and a phosphatase inhibitor cocktail (Sigma‐Aldrich). Immunoblotting was performed using antibodies against MIG‐6 (Cell Signaling Technology, Cat. #2440), GLUT1 (Abcam, clone SPM498), p‐EGFR (Tyr1173) (Cell Signaling Technology, clone 53A5), EGFR (Santa Cruz Biotechnology, clone 1005), pAKT(S473) (Cell Signaling Technology, Cat.9271), AKT (Cell Signaling Technology, clone C67E7), HIF1α (BD Biosciences, Clone 54), cMyc (Roche, clone 9E10), β‐actin (Sigma‐Aldrich, clone AC‐15), and α‐Tubulin (Sigma‐Aldrich, clone B‐5‐1‐2). Detailed antibody information is listed in Table EV4.

He J., Li C., Lee H., Shin D., Chern Y., Pereira De Carvalho B, & Chan C. (2021). MIG‐6 is essential for promoting glucose metabolic reprogramming and tumor growth in triple‐negative breast cancer. EMBO Reports, 22(5), e50781.

+ Open protocol

+ Expand

9

Protein Expression Analysis in Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein concentrations of cell or tumor lysates were measured by the BCA protein assay kit (Thermo Fisher Scientific, USA) and proteins were electrophoresed on SDS-PAGE gels. Separated proteins were then transferred onto PVDF membranes (Millipore, Bedford, MA, USA) followed by the incubation with specific antibodies against β-catenin (BD, Transduction Laboratories, KY, USA), c-myc (Roche Applied Sciences, Indianapolis, IN, USA), c-met (Santa Cruz, Biotechnology Inc., CA, USA), cyclin D1 (Santa Cruz Biotechnology), TC-1 (Santa Cruz Biotechnology), and β-actin (Sigma), and immunoblotted with the appropriate secondary antibody (Cell Signaling, Beverly, MA, USA). Chemiluminescent detection was performed using an ECL system (Amersham Pharmacia, Piscataway, NJ, USA).

Wu D., Li L, & Yan W. (2016). Knockdown of TC-1 enhances radiosensitivity of non-small cell lung cancer via the Wnt/β-catenin pathway. Biology Open, 5(4), 492-498.

+ Open protocol

+ Expand

10

Immunofluorescence Visualization of c-Myc

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunofluorescence slides were prepared according to the protocol of Audrey L. Atkin (Atkin, 1998) . Nuclear staining was performed with DAPI or Hoechst 33342. Primary antibodies were c-Myc (monoclonal, from mouse, by Roche) and c-Myc (polyclonal, from rabbit, by Santa Cruz). Secondary antibody was Alexa Fluor 555 (anti rabbit, life technologies).
Spheroblasting of cells was done with Zymolyase 100T (Seikagaku). Cells expressing GFPfused proteins were resuspended in PBS containing 4 % formaldehyde and dropped on polyline coated slides. After 5 minutes the slides were washed with PBS and the cells were coated with 10 µl Hoechst 33342 (5 µg/ml) and 10 µl Mounting Medium (according to Atkin, 1998) and coverslips.

Reinhardt A., Krutyhołowa R., Günzel C., Glatt S., & Breunig K.D. (2020). A double role of the Gal80 N-terminus in activation of transcription by Gal4p.

+ Open protocol

+ Expand

C myc

Lab products found in correlation

Close spelling variants of this product

10 protocols using c myc

Inhibition of ERK and c-MYC by ASP5878

Immunofluorescence Staining and Imaging Protocol

Western Blotting of Protein Samples

Western Blot Protein Extraction and Analysis

Analysis of Protein Interactions and Modifications

Immunofluorescence Staining of Transfected Cells

Western Blotting of Protein Samples

Western Blotting of Signaling Proteins

Protein Expression Analysis in Lysates

Immunofluorescence Visualization of c-Myc

About PubCompare

Ready to get started?