Does this result indicates enhanced proteasomal gedradation? - (Jul/31/2007 )
Dear All
My drug treatment downregulate a certain protein (target protein) in a cancer cell line, and this downregulation occurrs 1.5 hrs after drug treatment. So I did immuoprecipitation experiment to check wether my treatment increases the proteasomal degradation of that protein. In that experiment I pretreated the cells with MG132 (1000 nM) for 3 hrs followed by my drug treatment for 24 hrs, and in parallel I treated cells with MG 132 alone. After aliqouting 600 mcg of proteins, I then IP the target protein followed by western blotting using Ab of that target protein. I also run samples imuunoprecipitated with rabbit control IgG Ab. please check the blot in the attached file and let me know if this result indicates enhanced proteasomal gedradation or not?
thanks
well.... i would say no, as the band is sale intensity over the lanes, but a control would be nice (like actin for ex?)
you've mentionned your downregulation occurrs 1.5 hrs after drug treatment. So why don't you do time course ? or why 24h ? is there a possibility of adaptation ?
you don't very show down regulation by that mehcanism too
you've mentionned your downregulation occurrs 1.5 hrs after drug treatment. So why don't you do time course ? or why 24h ? is there a possibility of adaptation ?
you don't very show down regulation by that mehcanism too
Although band intensities of the target protein in all 3 lanes are similar, but as I said my treatment downregulates the expression of the target protein, so this means that pretreatment with MG132 corrected the induced downregulation of the target protein, so can we consider this correction as an indication of increased proterasomal degradatyion??
regarding your comment (but a control would be nice (like actin for ex?)), I only immunoprecipitated the target protein so the samples should not contain any other protein, so what do you mean by a control?
well how can you load your samples on the gel in order to make things comparable? 
if your treatment rescue the protein, it's not proteasomal increase. It may be blocking of the proteasmoe. If not your protein should have been more degraded.
But did you try proteasome inhibitors during assays without MG132 in order to be sure it's a proteasome issue ?
My drug treatment downregulate a certain protein (target protein) in a cancer cell line, and this downregulation occurrs 1.5 hrs after drug treatment. So I did immuoprecipitation experiment to check wether my treatment increases the proteasomal degradation of that protein. In that experiment I pretreated the cells with MG132 (1000 nM) for 3 hrs followed by my drug treatment for 24 hrs, and in parallel I treated cells with MG 132 alone. After aliqouting 600 mcg of proteins, I then IP the target protein followed by western blotting using Ab of that target protein. I also run samples imuunoprecipitated with rabbit control IgG Ab. please check the blot in the attached file and let me know if this result indicates enhanced proteasomal gedradation or not?
thanks
your question for proteolysis can better be discussed with total protein stain f.i. CBB or Ponceaus S; as Ab stain only selective it is difficult to distinguish between downregulation and proteolysis as a technical problem (during IP?); if you think of cellular proteolysis, you may check ubitinylation or SUMOylation with specific inhibitors
The attached file contains western blotting results after probing with ubiquitin Ab, do you think it provides any meaningful data?
Regarding your comment (your question for proteolysis can better be discussed with total protein stain f.i. CBB or Ponceaus S; as Ab stain only selective it is difficult to distinguish between downregulation and proteolysis as a technical problem (during IP?)), usually all lanes should show equal loading in case of gels prepared from whole cell lysate when they are stained with CBB, I want to know if the gel stained with CBB would look different if its there is increased proteolysis of a certain protein?
final question: if my results does not mean increased proteasomal degradation of the target protein, so how can explain the finding that pretreatment with proteasome inhibitors; MG132 and LLnL always correct drug treatment-induced downregulation of that protein? is it for example an upstream regulatory protein of that target protein that its proteasomal degradation is increased?
thanks
well... 1st, there is a thing i don't get. If your treatment by MG132 prevents degradation, it's proteasome inhibition are we right ?
second : a stain with ponceau should be done to see if there is degradation...
the ponceau is not specific to 1protein, so if ther's proteasome increased activity you may see stronger background (limited to a lane which discrimines from strong background of all membrane)
the experiment you're currently running can't say if you have proteasomal activation. It's strong possibility because a proteasome inhibitor treatment goes in same wa as MG132 treatment. But it's not a direct proof.
Proof may be reinforced by detectio of modification of your target protein.
So you may analyse (by mass spec for ex) if your protein gets ubiquitinylated and/or sumoylated
Radioactive pulse-chase experiment would be much cleaner way to show protein turnover rate.