WEEK SEVEN

August 10, 2017

WEEK SEVEN

- MONDAY 7/8/17 -

Seeded 4 x 6cm plates for transfection

This week, the plan is to repeat the experiment with the CD9/Tspan14 EC2 mutant to see if I get the same result for 9G1. I won't be using the CD9/Tspan14 var, as there was no recognition with any of the antibodies, and Ally mentioned in the lab meeting that most of the variable region is intracellular.

Split WT and Tspan14 over-expressing cells 1:5.

- TUESDAY 8/8/17 -

Today I transfected the cells with the appropriate plasmid DNA as follows:
1) pEF6.C-His
2) pEF6.C FLAG- hTspsn14
3) pEF6.C FLAG-hCD9
4) pEF6.C FLAG-hCD9/Tspan14EC2

-WEDNESDAY 9/8/17-

LAB MEETING NOTES:

(1) FACS data from experiment with Tspan14 cholesterol mutants.

The antibodies produced a more positive shift with the true mutant (Q to A). Dr Tomlinson pointed out that most other tetraspanins have Q/E at that position and perhaps the Q to E mutation makes Tspan14 bind cholesterol more tightly? We also thought that perhaps the Q to E mutant is more intracellular therefore has less recognition e.g. 1A3 doesn't recognise the mutant at all.

The rationale for this experiment was to determine whether 9G1 would specfically recognise the open conformation Tspan14 (as it previously didn't recognise the EC2), so it was interesting to see that all the antibodies recognised the mutant.

Dr Tomlinson and Connie have suggested I repeat this experiment as well as the one using pEF6 FLAG - hCD9/Tspan14 EC2 to see if 9G1 still behaves the same way.

MICROSCOPY IMAGES:

The microscopy images (see Week 6-Tues) shows punctate staining with the cells stably transfected with hTspan14 and no fluorescence signal with the WT cells, which suggests the protein might be mostly intracellular. I will image the WT cells again at a higher intensity tomorrow to see if there is more fluorescence signal.

Dr Tomlinson mentioned that it would be useful in future to do surface biotinylation experiments to quantify the surface expression of Tspan14 on HEK cells.

After the lab meeting, I split the WT cells 1:5 and the HEK cells stably transfected with Tspan14. Connie also split a plate of HEK ADAM10 knock-out cells, so I can do another FACS experiment on Friday.

- THURSDAY 10/8/17 -

Today I imaged the HEK WT cells again with the confocal microscope at 16% intensity and took z-stacks of 0.5 um. Comparing the images to the control shows that some of the antibodies (1A3, 6E5, 10F3) produce a stronger signal than the background noise seen in MOPC control. This could suggest that they are recognising endogenous Tspan14 on the cells.

- FRIDAY 11/8/17 -

We had discussed in the lab meeting that if endogenous Tspan14 is interacting with ADAM10, the ADAM10 could be blocking the epitopes for the antibody to bind. Today I did a FACS experiment using HEK ADAM 10 KO cells and I used HEK cells stably transfected with Tspan14 as the positive control to see if this is the case.

Unfortunately, the data was disappointing and rather confusing. None of the antibodies seemed to recognise Tspan14 on the KO cells, which was unexpected. Although there is a small positive shift with 6E5 and 10F3, which could suggest that they're recognising an epitope masked by ADAM10. I will redo this experiment next week to see if this repeats.

The data with the stably transfected cells shows most of the antibodies do recognise overexpressed Tspan14, however the shifts are a lot weaker than what I've been seeing in previous experiments with hTspan14 transfected cells. 1A3 and 3G3 didn't produce strong positive shifts as seen previously :(