1. - + PKA A B Purified LASP-1 kDa LASP pLASP dpLASP 72 LASP 55 pLASP-14640 33
dpLASP-146 25
PKA 17
Control
Forskolin
Thrombin
Control
Forskolin
Thrombin
Control
Forskolin
Thrombin
Figure S1. Specificity of the antibodies recognizing phosphorylated pLASP-1 and dephosphorylated dpLASP-1 at position 146. Specificity was tested in Western Blots with (A) recombinant LASP-1 phosphorylated at Ser-146 with purified catalytic subunit of PKA (Butt et al., JBC, 2003) and with (B) washed human platelets stimulated with either forskolin (PKA activation, Ser-146 phosphorylation) or thrombin (Src kinase activation, Tyr-171 phosphorylation) (Traenka et al., TH, 2009).
(A) The newly developed antibody pLASP-146 does not recognize LASP-1 in its unphosphorylated state or after tyrosine phosphorylation while a strong signal appears after LASP-1 is phosphorylated by PKA. Concurrently, the signal for dpLASP-146 decreases.
(B) pLASP-146 and dpLASP-146 antibodies are highly specific and detect only one specific band at the expected size around 36 kDa in intact human platelets. In the non-phosphorylated probes LASP-1 staining with dpLASP-146 antibody almost completely overlaps with the staining of the polyclonal LASP-1 antibody.