Yesteday,

1.I completed the western blot of BL treated gul4 and Ws-2.  The results is similar with last time. The gul4 shows slightly increased level of non phosphorylated BES1. The coomasie blue staining shows roughly the equal loading but the with anti Actin gul4 sample show  thick bands similar with the last time. I am wandering is it due to the over expression of actin in gul4 with long hypocotyl? or something else. This time I did measure the protein level with 3 replicates of each samples and calculated average for loading.Please find the attached file for the results.

2. I counted the no. of seedlings in T2 of OX lines CYP83B1 in Ws-2 and gul4 backgraounds as I mentioned yesterday.

 And collected the tissue for Western. Regarding the lines which have WT phenotype and gul4 phenotype I collected the tissue seperately.Is it necessary to do western seperately for WT phenotype and gul4 phenotype within the single line to corelate the expression level of protein with phenotype?

3. Culture the expression clone of 35S:gul4 CDS:YFP:HA.

Today

1. The first set of seedlings growing for microarray samples is nine day in growth room today. So I will collect tissues today.

2. I will do BL treatment (concentration-0,10-9,10-8, 10-7, 10-6 M) of gul4 and Ws-2 in liquid MS without sugar for 2 hours as before and will collect the tissue for RT PCR to see the expression level of BR related genes and CYP83B1 and related genes  and western to see the BES1 state. This experiment is to reconfirm the 1st time results found in 10-8 and a0-6 M  BL concentration treatments.

3. I could not complete the yesterday's plan experiment, so today I willl continue those experiments.