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Hap030098

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HAP030098 is a laboratory instrument designed for conducting research and analysis in various scientific fields. It is a multi-purpose equipment that can be utilized for a range of applications, but a detailed description of its core function is not available at this time.

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Lab products found in correlation

Sc 101199, by Santa Cruz Biotechnology (5 mentions) Yap antibody, by Santa Cruz Biotechnology (1 mentions) Fluorescence secondary antibody, by Thermo Fisher Scientific (1 mentions) Fitc conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions) Alexa flour 647, by Thermo Fisher Scientific (1 mentions) Oil na1.4 objective, by Nikon (1 mentions) Ab9106, by Abcam (1 mentions) Gb12001, by Wuhan Servicebio Technology (1 mentions)

5 protocols using hap030098

1

Immunoprecipitation of RNF187 and YAP

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Immunoprecipitation was performed as described in the previous study. The BT549 cells total cell lysls were pre-cleared with rabbit IgG for 2 h and subsequently immunoprecipitated with RNF187 antibody (HAP030098, Sigma, 1/200) or YAP antibody (SC101199, santa cruz, 1/200) over night, while rabbit IgG (Santa Cruz) was used as the negative control. The bounded protein was analyzed by Anti-YAP antibody (SC-101199, Santa Cruz; 1/1000) or RNF187 antibody (HAP030098, Sigma, 1/1000).
Wang Z., Kong Q., Su P., Duan M., Xue M., Li X., Tang J., Gao Z., Wang B., Li Z., Liu Y., Yang X., Cao R., Song T., Wang K., Cai Y., Wu D., Li J., Wu G., Guled A.M., Zhu J., Yan C, & Zhuang T. (2020). Regulation of Hippo signaling and triple negative breast cancer progression by an ubiquitin ligase RNF187. Oncogenesis, 9(3), 36.
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2

Immunofluorescence Staining of RACO-1 and YAP

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EC9706 cells were fixed with 4% paraformaldehyde for 10 minutes, 0.5% Triton perforated for 5 minutes, after that rinsed with PBS for three times and then sealed with 5% BSA at room temperature for 1.5 hours. Rabbit anti‐RACO‐1 polyclonal antibody (HAP030098, Sigma) and mouse anti‐YAP monoclonal antibodies (SC‐101199, Santa Cruz) were used to incubated and then combined with fluorescence secondary antibody (Invitrogen). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. After stained with DAPI, images were observed under laser scanning confocal microscopy (Nikon C2+/si+ Japan).
Pang D., Wang W., Zhou X., Lu K., Zhang J., Chen Z., Wang L., Shen F., Chen Z., Wang S., Hou J., Zhang A., Lv B., Gao C., Yan Z., Hu Y., Chang T., Wang L, & Li X. (2020). RACO‐1 modulates Hippo signalling in oesophageal squamous cell carcinoma. Journal of Cellular and Molecular Medicine, 24(20), 11912-11921.
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3

Immunofluorescence Imaging of RNF187 and YAP

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EC109 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-RNF187 polyclonal antibody (HAP030098, Sigma, 1/50) and mouse anti-YAP monoclonal antibodies (SC-101199, Santa Cruz, 1/50) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60× oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
Wang Z., Kong Q., Su P., Duan M., Xue M., Li X., Tang J., Gao Z., Wang B., Li Z., Liu Y., Yang X., Cao R., Song T., Wang K., Cai Y., Wu D., Li J., Wu G., Guled A.M., Zhu J., Yan C, & Zhuang T. (2020). Regulation of Hippo signaling and triple negative breast cancer progression by an ubiquitin ligase RNF187. Oncogenesis, 9(3), 36.
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4

Immunohistochemistry of Triple-Negative Breast Cancer

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Forty triple negative breast cancer samples were collected from QIlu Hospital of Shandong University. All the triple negative breast cancer samples were examined in ER, PR, and HER2 status. The immuno-histochemisty of RNF187 and YAP were carried out according to standard method. The IHC results of RNF187 and YAP were examined through pathological specialists. The rabbit anti-RNF187 polyclonal antibody (HAP030098, Sigma, 1:100) and mouse anti-YAP monoclonal antibodies (SC-101199, Santa Cruz, 1/100) were used for IHC analysis. The size of the FFPE section was prepared in 4 μm. The results of YAP/RNF187 staining were determined by two independent certified pathologists.
Wang Z., Kong Q., Su P., Duan M., Xue M., Li X., Tang J., Gao Z., Wang B., Li Z., Liu Y., Yang X., Cao R., Song T., Wang K., Cai Y., Wu D., Li J., Wu G., Guled A.M., Zhu J., Yan C, & Zhuang T. (2020). Regulation of Hippo signaling and triple negative breast cancer progression by an ubiquitin ligase RNF187. Oncogenesis, 9(3), 36.
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5

Western Blot Analysis of Cellular Proteins

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Cellular proteins were extracted by RIPA buffer. Proteins were isolated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was sealed at room temperature for 2 hours and then incubated with a primary antibody at room temperature for 2 hours or 4° overnight.
The antibodies used in this study were listed here: Anti‐RACO‐1 (HAP030098, Sigma); Anti‐YAP (SC‐101199, Santa Cruz); Anti‐HA (AB0004, Abways); Anti‐myc (ab9106, Abcam); and Anti‐Actin (GB12001, Servicebio). Membranes were washed and then incubated in secondary antibodies peroxidase‐conjugated AffiniPure Goat Anti‐Mouse IgG or Goat Anti‐Rabbit IgG. Enhanced chemiluminescence system was used to observe the cell membrane.
Pang D., Wang W., Zhou X., Lu K., Zhang J., Chen Z., Wang L., Shen F., Chen Z., Wang S., Hou J., Zhang A., Lv B., Gao C., Yan Z., Hu Y., Chang T., Wang L, & Li X. (2020). RACO‐1 modulates Hippo signalling in oesophageal squamous cell carcinoma. Journal of Cellular and Molecular Medicine, 24(20), 11912-11921.
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