Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

摘要

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

摘要

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

引言

Disruption of MST1/Hippo signaling has been connected to developmental disorders and carcinogenesis¹. In mammals, the kinases MST1 and MST2 activate (phosphorylate) MOB1 and LATS1/2, the latter of which then phosphorylates and inactivates the transcriptional co-activator Yes-associated protein (YAP)². In its active (unphosphorylated) form, YAP has oncogenic activity, enhancing transcription of cell proliferation genes; conversely, when YAP is inactivated by the Hippo pathway, cell proliferation is suppressed and apoptosis promoted³. In tissues, MST1 and MST2 exist mainly as active homodimers, but ....

方案

1. Preparation of Solutions

1. Prepare fixative solution: 4% paraformaldehyde (PFA) in 1x PBS. For 10 mL, take 2.5 mL of 16% PFA and add 7.5 mL of 1x PBS.
   Hazards: PFA is carcinogenic at low doses. Fumes and skin contact are hazardous. Store at -20 °C.
2. Prepare permeabilization solution: 0.1% Triton X-100 in 1x PBS. For 100 mL of solution, add 100 µL of Triton X-100 into 100 mL of 1x PBS. Store at room temperature (RT).
3. Prepare Wash Buffer: 1x TBST. For 1 L, take 100 mL of 10x TBS, 890 mL of dH₂O, and 10 mL of Tween 20 (10%).
4. Prepare blocking solution as supplied by kit. Alternatively, ....

结果

We used the PLA assay to test the interaction between MST1 and MST2 in HEK-293 and iHSC. The cells were fixed, permeabilized, and stained with various antibodies, followed by in situ amplification according to the PLA protocol (Figure 1). To document the level of MST1/MST2 heterodimerization, cells were stained with MST1 and MST2 antibodies (Figure 1A, 1C, and 1G). As a positive control, we also used ERK.......

讨论

We found it useful to use glass chamber slides for this experiment, as it is very convenient to perform experiment with several (14-16) cell lines and there is no need to change the sample every time during microscopy analysis. A few complications may arise, such as an increased risk for cross-contamination with antibodies. Therefore, we suggest washing every well individually instead of using a Coplin jar, despite the increased duration of the experiment. In addition, removal of silicone insert is a delicate affair and .......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
载玻片	Thermo Fisher Scientific	178599	16 孔载玻片，载玻片
PLA 探针 抗小鼠 Minus	Sigma-Aldrich	DUO92004	包含 1x 封闭溶液、1x 抗体稀释剂
PLA 探针 Anti-Rabbit PLUS	Sigma-Aldrich	DUO92002	包含 1x 封闭溶液、1x 抗体稀释剂
洗涤缓冲液、Flurescence	Sigma-Aldrich	DUO82049	包含洗涤缓冲液 A 和洗涤缓冲液 B
封固剂以及 DAPI	Sigma-Aldrich	DUO82040
检测试剂 Red	Sigma-Aldrich	DUO92008	包含 5x 连接、1x 连接酶、5x 稀释红、1x 聚合酶
p44/42 MAPK （erk1/2） 抗体	细胞信号传导	9102s
磷酸化 p44/42 MAPK （erk1） （Tyr204）/（errk2） （Tyr187）	细胞信号转导	5726s	pERK 抗体
MST2 抗体	细胞信号转导	3952s
Krs-2 （RJ-5）	Santa Cruz	sc-100449	MST1 抗体
16% 多聚甲醛	电子显微镜科学	15710	在 PBS 中稀释至 4% PFA 用于固定溶液
Triton x100	Fisher BioReagents	BP 151-500	在 1xPBS 中制备 0.1% Triton x-100 用于透化溶液
聚焦显微镜	Leica TCS SP8，	使用 ImageJ 软件进行