For target pathway-specific activation on plasma membrane Plasma membrane-specific protein translocation platform
SLIPT-PM

To establish SLIPT-PM experiments, you need not only our products but also ^iK6DHFR-expression plasmids. Before order of our SLIPT-PM product, please read following processes.

^iK6DHFR-expression plasmids containing a multi-cloning site and EGFP gene are distributed by plasmid bank addgene (Table 1). You can also construct plasmids from eDHFR^WT gene by yourself, according to the reference paper (Ref. 3). Plasmids for control experiments mentioned in the “SLIPT-PM Experimental Guide Book” are also distributed by addgene (Table 2)

※ Please find each application note in the “SLIPT-PM Experimental Guide Book”.

You can see the technology background, principle and guides for experimental setting in our “SLIPT-PM Experimental Guide Book” below URL. If you are interested in SLIPT-PM experiments more detail, please find it prefer to order of our product. The other application notes are also described in the “SLIPT-PM Experimental Guide Book”.
Download “SLIPT-PM Experimental Guide Book” here

■Target signaling pathway: cRaf → MEK → ERK
■Constructs in the experiment:
- ^iK6DHFR-fused protein: EGFP-^iK6DHFR-cRaf^*1 （addgene ID：172107）
^*1In this experiment, cRaf indicates the full-length cRaf protein
- Reporter protein: Nucleus ERK activity sensor (^ERKKTR^*2-mKusabiraOrange (mKO))
^{*2 ERK}KTR is kinase translocation reporter for ERK
■SLIPT-PM concentration: SLIPT-PM 5 μM
■Cells: HeLa cells
■Reaction medium: serum-free DMEM
■Experimental design:
cRaf, a downstream factor of Ras, can be activated by Ras on the PM and subsequently activate MEK-ERK pathway. Phosphorylated ERK is subsequently translocated into the nucleus and activates further downstream transcription factors. In this model experiment, ^iK6DHFR-fused cRaf protein (EGFP-^iK6DHFR-cRaf) is translocated to the PM by SLIPT-PM and promotes activation of MEK-ERK pathway on the PM without Ras activity. ERK activity is monitored by a nucleus ERK activity sensor (^ERKKTR-mKO).

Before the addition of SLIPT-PM, EGFP-^iK6DHFR-cRaf and ^ERKKTR-mKO were constantly localized in cytosol and mainly nucleus, respectively. By the addition of SLIPT-PM, at first, EGFP-^iK6DHFR-cRaf rapidly translocated to the PM (t_1/2~1 min) and subsequent elimination of nuclear ^ERKKTR-mKO to cytosol was observed. In the presence of MEK inhibitor, although EGFP-^iK6DHFR-cRaf translocated to the PM, ^ERKKTR-mKO localization was not changed. This result indicates SLIPT-PM promotes membrane-binding of EGFP-^iK6DHFR-cRaf and EGFP-^iK6DHFR-cRaf activates the MEK-ERK signaling pathway.

■Target signaling pathway: cRaf → MEK → ERK
■Constructs in the experiment:
- ^iK6-DHFR-fused protein: cRaf^*1-mNeonGreen (mNG) -^iK6DHFR（addgene ID：172107）
^*1 In this experiment, cRaf indicates the full-length cRaf protein
- Reporter protein: mCherry-ERK
■SLIPT-PM concentration: 5 μM
■Free-TMP concentration: 50 μM
■Cells: HeLa cells
■Reaction medium: serum-free DMEM
■Experimental design:
cRaf, a downstream factor of Ras, can be activated by Ras on the PM and subsequently activate MEK-ERK pathway. Phosphorylated ERK is translocated into the nucleus and activates further downstream transcription factors. In this model experiment, ^iK6DHFR-fused cRaf protein (cRaf-mNG-^iK6DHFR) is translocated to the PM by SLIPT-PM and promotes activation of MEK-ERK pathway. Nuclear translocation of mCherry-ERK is monitored as a sensor of MEK-ERK pathway. After then, by addition of Free-TMP induced release of cRaf-mNG-^iK6DHFR from the PM to cytosol. Furthermore, wash out of Free-TMP reproduces PM-recruitment of cRaf-mNG-^iK6DHFR by remaining SLIPT-PM on the PM again and reactivates MEK-ERK pathway.

Before the addition of SLIPT-PM, both cRaf-mNG-^iK6DHFR and mCherry-ERK constantly localized in cytosol. By the addition of SLIPT-PM, firstly, cRaf-mNG-^iK6DHFR translocated to the PM (t_1/2~7 min) and subsequently, nuclear translocation of mCherry-ERK was observed. The addition of Free-TMP at 20 min clearly canceled the PM-tethering of cRaf-mNG-^iK6DHFR and nuclear localization of mCherry-ERK was decreased. The cell medium containing Free-TMP was continuously exchanged using a peristaltic pump from 40 min to 60 min recovered PM-translocation of cRaf-mNG-^iK6DHFR and nuclear localization of mCherry-ERK again without the addition of SLIPT-PM. In this experiment, reversible regulation of cRaf-mNG-^iK6DHFR and mCherry-ERK was successfully observed at least two cycles of Free-TMP addition and medium change. This result indicates SLIPT-PM is still tethering on the PM after Free-TMP addition and medium change.

Scale bar 10 μm