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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Mol Cell Neurosci. 2009 Nov 10;43(2):188. doi: 10.1016/j.mcn.2009.10.009

Figure 2. Biochemical association of PDLIM5 with SPAR.

Figure 2

(A) Adult rat brains were homogenized, subjected to biochemical fractionation, and immunoblotted for PDLIM5 (GU8 rabbit antibodies), synaptophysin (syn), or PSD-95 as indicated. P1, crude pellet; P2, crude synaptosomes; P3, light membrane; S3, cytosolic; LP1, synaptic membrane; LP2, synaptic vesicle; PSD, postsynaptic density. PDLIM5 co-fractionated with both pre- and postsynaptic markers. COS-7 lysates expressing recombinant PDLIM5a or PDLIM5b are included (left lanes) to show the expected sizes of isoforms. (B) Cultured hippocampal neuron lysates (hpc) were harvested at various days in vitro (DIV) and immunoblotted for PDLIM5 (a and b isoforms are indicated). Beta actin levels are shown as loading control. To show the expected size of PDLIM5 and the specificity of antibodies, whole brain lysates or COS-7 transfected cell lysates of myc-Enigma or HA-PDLIM5a are included (left lanes as indicated) and immunoblotted with antibodies shown at bottom of blots. Note recombinant myc-Enigma and HA-PDLIM5a migrate slightly higher than endogenous counterparts due to the epitope tag. Unxfect, untransfected. (C) Adult rat brain lysates were immunoprecipitated with antibodies against PDLIM5 (GU8 rabbit) or SPAR rabbit, or nonimmune rabbit IgG, and immunoblotted for endogenous SPAR and PDLIM5, as indicated. COS-7 lysates expressing recombinant SPAR or PDLIM5 are included (left lanes) to show the expected size of full length proteins. Inset at top shows a longer exposure of a SPAR western from a duplicate experiment showing weak but detectable SPAR coimmunoprecipitated with PDLIM5 antibodies. IN, 10% lysate used per IP (1% lysate for overexposed inset). (D) Rat brain lysates immunoprecipitated with two independent anti-SPAR guinea pig sera (gp1 and gp2), or preimmune sera from the same animals (preimm1 and preimm2). Precipitates were immunoblotted for SPAR or PDLIM5, as indicated. Asterisk represents IgG heavy chain. IN, 10% of input used per immunoprecipitation reaction. (E) COS-7 cells were transfected with myc-tagged Act2 domain, HA-tagged full-length PDLIM5a or PDLIM5b, or empty mycGW1 vector as indicated. Lysates were immunoprecipitated with myc agarose beads and immunoblotted with HA epitope antibodies. Input is shown demonstrating equal expression of PDLIM5 between conditions. All molecular weights shown in kilodaltons.