All lanes : Anti-ZFP36L1 (phospho S92) antibody [EPR19926] (ab204922) at 1/2500 dilution

Lane 1 : Unstimulated wild-type mouse CD4+ and CD8+ T cells, whole cell lysate (Untreated membrane)
Lane 2 : Wild-type mouse CD4+ and CD8+ T cells treated with 10ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1mM Ionomycin for 3 hours, whole cell lysate (Untreated membrane)
Lane 3 : Unstimulated ZFP36L1 knockout mouse CD4+ and CD8+ T cells, whole cell lysate (Untreated membrane)
Lane 4 : ZFP36L1 knockout mouse CD4+ and CD8+ T cells treated with 10ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1mM Ionomycin for 3 hours, whole cell lysate (Untreated membrane)
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate (Untreated membrane)
Lane 6 : Unstimulated wild-type mouse CD4+ and CD8+ T cells, whole cell lysate (Phosphatase treated membrane)
Lane 7 : Wild-type mouse CD4+ and CD8+ T cells treated with 10ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1mM Ionomycin for 3 hours, whole cell lysate (Phosphatase treated membrane)
Lane 8 : Unstimulated ZFP36L1 knockout mouse CD4+ and CD8+ T cells, whole cell lysate (Phosphatase treated membrane)
Lane 9 : ZFP36L1 knockout mouse CD4+ and CD8+ T cells treated with 10ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1mM Ionomycin for 3 hours, whole cell lysate (Phosphatase treated membrane)
Lane 10 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate, (Phosphatase treated membrane)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/15000 dilution (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773); and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776))

Predicted band size: 36 kDa
Observed band size: 36-47 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 3% NFDM/TBST.

Lysates from the ZFP36L1fl/fl CD4 cre KO mouse were kindly provided by Dr Fiamma Salerno, Turner Lab, Babraham Institute. 

Lysates used for the WB were isolated by negative selection using biotinylated abs against Ter119, CD11b, CD11c, Gr1, CD19, B220, F4/80 and NK1.1 to leave a population of CD4 and CD8 positive T cells (the purity of the population was >95% as checked by flow). 

Mouse anti-vinculin ab130007, used as the loading control.   

All lanes : Anti-ZFP36L1 (phospho S92) antibody [EPR19926] (ab204922) at 1/5000 dilution

Lane 1 : HEK-293T (human embryonic kidney epithelial cell) transfected with an empty vector (vector control) containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with human ZFP36L1 expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T transfected with human ZFP36L1 expression vector containing a myc-His-tag®, then treated with alkaline phosphatase for 1 hour on the membrane
Lane 4 : HEK-293T transfected with human ZFP36L1 S92A mutant expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 36-47 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 2% BSA/TBST.

All plasmids were kindly provided by Dr Fiamma Salerno, Turner Lab, Babraham Institute. 

The expression profile observed is consistent with what has been described in the literature (PMID: 17030608). 

Exposure time: 8 seconds

Dot blot analysis of ZFP36L1 (phospho S92) labeled with ab204922 at 1/1000 dilution.

Lane 1: ZFP36L1  (phospho S92) peptide (aa87-97).
Lane 2: ZFP36L1 non-phospho peptide (aa87-97).
Lane 3: ZFP36L1 (phospho S125) peptide (aa120-130).
Lane 4: ZFP36L1  non-phospho peptide (aa120-130).

Blocking and dilution buffer: 5% NFDM/TBST.Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Exposure time: 3 minutes.