Recombinant TLRR does not affect the phosphatase activity of PP1 isoforms.

<p>The activity of His6 tagged PP1 isoforms alone (red bars) or with an equal amount of TLRR (blue bars) was assayed for PP1-specific activity as described in the text using the RediPlate 96 EnzChek® Serine/Threonine Phosphatase Assay kit from Invitrogen.</p

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids-0

Leucine-rich repeats (LRRs) are indicated with solid underlining and the peptide used for preparation of antibody indicated with dashed underlining. The putative PP1 binding site is boxed. (B) The TLRR repeats are aligned with the LRR consensus sequence for the sds22 subfamily of LRR proteins. (C) An alignment between TLRR and Ppp1r7 is shown with the LRRs indicated with overlines (TLRR) and underlines (Ppp1r7). Multiple sequence alignment was done using ClustalW 1.82 and the results displayed using BOXSHADE 3.31. LRRs in TLRR and Ppp1r7 were identified using the Pfam protein domain database [35].<p><b>Copyright information:</b></p><p>Taken from "Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids"</p><p>http://www.biomedcentral.com/1471-2121/9/9</p><p>BMC Cell Biology 2008;9():9-9.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2270827.</p><p></p

The expression profiles of TLRR and PP1γ2 are conversely related during development of the testis.

<p>50 µg testis lysate from animals at the indicated days after birth was separated by SDS-PAGE, transferred to membrane, and probed with antibodies specific for the indicated proteins.</p

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids-4

D DAPI (blue, nuclei) as described in the Methods section. (A'-D') TLRR signal corresponding to each of the merged images shown in A-D. Increasingly elongated spermatids are shown from panels A, A' through D, D'. TLRR is associated with the manchette in mid stage spermatids (arrowheads, panels C and C') and this staining narrows with the elongation of the manchette in approximately step 15 spermatids (arrowheads, panels D and D'). (E, E') Negative control for these experiments where TLRR specific primary antibody was omitted. Bar represents 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids"</p><p>http://www.biomedcentral.com/1471-2121/9/9</p><p>BMC Cell Biology 2008;9():9-9.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2270827.</p><p></p

Phosphatase activity is associated with the TLRR complex immunoprecipitated from adult testis lysate.

<p>TLRR complex phosphatase activity was measured using the EnzChek® Phophatase Assay Kit (Invitrogen) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021767#s4" target="_blank">Materials and Methods</a>. Either TLRR antibody or Normal Rabbit IgG (NRIgG) as negative control were linked to Sepharose beads and incubated with 1 mg (25 µl of 40 µg/µl) CD-1 adult mouse testis lysate. Proteins bound to beads were eluted and added to fluorescence substrate and resultant fluorescence was measured after 60 min. The results are significant at p = 0.01.</p

TLRR-associated phosphatase activity varies with developmental stage of the testis.

<p>(A) Testis lysate from day 7, 14, 21, 35, or 56 mice was incubated with Sepharose beads linked to TLRR antibody. Protein phosphatase activity of bound proteins was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021767#pone-0021767-g001" target="_blank">Figure 1</a> using the Enzchek® assay for total phosphatase activity and the results are displayed relative to the activity at 56 days (set as 100%). Protein phosphatase activity at day 7 and day 21 are significantly different at p = 0.05. (B) Testis lysate from day 7, 14, 21, 35, or 56 mice was immunoprecipitated with TLRR antibody beads and the PP1-specific phosphatase activity in bound proteins was measured with Rediplate 96 EnzChek® Serine/Threonine Phosphatase Assay kit (Invitrogen). Fluorescence was measured at 460 nm. PP1 activity is significantly different between day 7 and day 21 (asterisks) and between day 21 and day 35 (deltas). Differences are significant at p = 0.05.</p

TLRR, kinesin-1B and tubulin are phosphorylated during the first round of spermatogenesis in the developing testis.

<p>2 mg testis lysate from animals at the indicated days after birth was immunoprecipitated with a mixture of anti-phosphoserine antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021767#s4" target="_blank">Materials and Methods</a>. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to membrane and blotted with antibodies specific to the proteins indicated to the right. –C indicates proteins immunoprecipitated with normal rabbit IgG.</p

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids-2

E LRR domains are indicated with black boxes and below are shown the regions tested for interaction with the KIFC1 bait plasmid and the results of interaction tests. (B) Plate assay of interaction of TLRR deletion constructs with KIFC1bait. The plate on the left selects only for yeast harboring both bait and prey plasmids; all cotransformants are able to grow. The plate on the right also lacks adenine and histidine and is therefore selective for transactivation of the reporter genes. Transactivation of the MEL1 reporter gene is detected by incorporation of X-α-gal into the media resulting in blue colonies. Positive control for protein interaction (+C) is yeast cotransformed with pGADT7-T and pGBKT7-53 while cells containing pGADT7 and pGBKT7-Lam (-C) is negative control. (C) Liquid assay of interaction of TLRR deletion constructs with KIFC1bait. The β-galactosidase activity of cultures of TLRR/KIFC1 bait cotransformants was determined as described in Methods. Each assay was repeated at least three times. Standard error of the mean for each transformant is indicated by bracketed lines in panel C.<p><b>Copyright information:</b></p><p>Taken from "Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids"</p><p>http://www.biomedcentral.com/1471-2121/9/9</p><p>BMC Cell Biology 2008;9():9-9.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2270827.</p><p></p

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids-6

Leucine-rich repeats (LRRs) are indicated with solid underlining and the peptide used for preparation of antibody indicated with dashed underlining. The putative PP1 binding site is boxed. (B) The TLRR repeats are aligned with the LRR consensus sequence for the sds22 subfamily of LRR proteins. (C) An alignment between TLRR and Ppp1r7 is shown with the LRRs indicated with overlines (TLRR) and underlines (Ppp1r7). Multiple sequence alignment was done using ClustalW 1.82 and the results displayed using BOXSHADE 3.31. LRRs in TLRR and Ppp1r7 were identified using the Pfam protein domain database [35].<p><b>Copyright information:</b></p><p>Taken from "Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids"</p><p>http://www.biomedcentral.com/1471-2121/9/9</p><p>BMC Cell Biology 2008;9():9-9.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2270827.</p><p></p

The quantity of TLRR-PP1 complex varies with developmental age of the testis.

<p>2 mg testis lysate from animals at the indicated days after birth was immunoprecipitated with either the pan-PP1 antibody FL-18 (A) or an antibody specific to the gamma 2 isoform (B). Bound proteins were separated by SDS-PAGE, transferred to membrane and probed with the corresponding antibody or an antibody to TLRR. In the negative control (-C) normal rabbit IgG was used in place of the PP1 antibody.</p