All lanes : Anti-TrkB (phospho Y705) antibody [EPR22298-67] (ab229908) at 1/1000 dilution

Lane 1 : Human brain
Lane 2 : Human Hippocampus
Lane 3 : Human cerebellum
Lane 4 : Human spinal cord

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 92 kDa


Exposure time: 5 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Observed MW: 140 kDA

All lanes : Anti-TrkB (phospho Y705) antibody [EPR22298-67] (ab229908) at 1/1000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Mouse hippocampus
Lane 3 : Mouse P7 thalamus
Lane 4 : Mouse cerebellum
Lane 5 : Rat brain
Lane 6 : Rat hippocampus
Lane 7 : Rat P7 thalamus
Lane 8 : Rat cerebellum
Lane 9 : Mouse P5 cerebral cortex
Lane 10 : Mouse P3 retina
Lane 11 : Mouse P20 retina
Lane 12 : Rat spinal cord

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 92 kDa


Exposure time: 180 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Observed MW: 140 kDA

All lanes : Anti-TrkB (phospho Y705) antibody [EPR22298-67] (ab229908) at 1/1000 dilution

Lane 1 : Untreated PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg
Lane 2 : PC-12 treated with 100ng/ml NGF (ab9796) for 5 minutes at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 92 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The phosphorylation sites are conserved between TrkA and TrkB: Tyr674/675 of TrkA corresponds to Tyr706/707 in TrkB. Therefore, the TrkB (phospho T705) antibody can detect the phosphorylation of TrkA T674/675 in PC-12 cells treated with NGF. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 23951412).

  Exposure time: 3 minutes

All lanes : Anti-TrkB (phospho Y705) antibody [EPR22298-67] (ab229908) at 1/5000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with human TrkB (WT) expression vector containing a myc-His-tag®, whole cell lysate (Untreated membrane)
Lane 3 : HEK-293T transfected with human TrkB (WT) expression vector containing a myc-His-tag®, whole cell lysate (Phosphatase treated membrane)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 92 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 24959744). Besides, TrkB auto-phosphorylation was observed when TrkB was over-expressed in HEK293 cells, which has been reported in the literature (PMID: 25632160). 

Exposure time: 7 seconds

All lanes : Anti-TrkB (phospho Y705) antibody [EPR22298-67] (ab229908) at 1/5000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with mouse TrkB (WT) expression vector containing a myc-His-tag®, starved for 6 hours, whole cell lysate
Lane 3 : HEK-293T transfected with mouse TrkB (WT) expression vector containing a myc-His-tag®, starved for 6 hours, then treated with 50ng/ml BDNF (ab9794) for 10 minutes, whole cell lysate (Untreated membrane)
Lane 4 : HEK-293T transfected with mouse TrkB (WT) expression vector containing a myc-His-tag®, starved for 6 hours, then treated with 50ng/ml BDNF (ab9794) for 10 minutes, whole cell lysate (Phosphatase treated membrane)
Lane 5 : HEK-293T transfected with mouse TrkB (mutant Y705A) 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 1/50000 dilution

Predicted band size: 92 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 24959744). Besides, TrkB auto-phosphorylation was observed when TrkB was over-expressed in HEK293 cells, which has been reported in the literature (PMID: 25632160). 

Exposure time: 48 seconds

Dot blot analysis using ab229908 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Lane 1: TrkB (phospho T705) peptide (aa697-708)

Lane 2: TrkB (phospho T705) peptide (aa702-712)

Lane 3: TrkB non-phospho peptide (aa697-712)

Blocking/diluting buffer and concentration: 5% NFDM/TBST