Hi everyone,

I'm trying now for quite some time to express human kindlin2 in HEK cells and I'm having a hard time. I amplified kindlin cDNA (accession number: BG330159 ) and cloned it in pEGFP-C1 (invitrogen) using XhoI and BamHI restriction sides (as reported in Theodosiu et al eLife 2016). Control restriction digest and sequencing results were perfectly fine. Gene is in frame. However, there is no expression in HEK cells. I even analyzed the pellet and supernatant after lysate clearing. I even cooked the cells immediately in Laemmli buffer. Kindlin is nowhere to be found. I cloned it with different tags (mCherry, myc and NEF) and never get a signal with Western blotting. Other proteins (e.g paxillin) are all nicely expressed using the same vectors and cloning strategies.

Does anyone have a logic explanation for this? Or had similar problems? What's the magic? or am I missing the obvious?

Your help and suggestions would be much appreciated!

Thank you very much in advance