Journal: FASEB BioAdvances

Article Title: In search of alternatively spliced alpha‐Klotho Kl1 protein in mouse brain

doi: 10.1096/fba.2020-00066

Figure Lengend Snippet: Model of spliced Kl1 αKlotho and characterization of antibodies. A. Model of spliced αKlotho transcript and the hypothetical spliced Kl1 αKlotho protein (spKl1). Fifteen amino acid for spKl1‐specific epitope (spE15) was used to generate antibody against spKl1. B. Left: myc‐tagged Kl1 and myc‐tagged spKl1 were expressed in HEK293 cells and immunoblotted with anti‐KL1, anti‐myc, and anti‐spE15 (raised against the 15 amino acid unique to spKl1). Right: primary culture of human choroid plexus cells (HCP) were transfected with full‐length αKlotho (KL), Kl1, spliced Kl1 (spKl1), and vector only, and cell lysate were immunoblotted with anti‐Kl1 or anti‐spE15. C. Immunocytochemistry of HEK293 cells transfected with spKl1 using anti‐Kl1 (left) or anti‐spE15 (middle). Merged image is on the right. D. Epitope mapping of rat anti‐KL1 and rat anti‐KL2. Top panel shows the location of the myc‐tagged peptides A to F in relation to full‐length αKlotho. Middle panel show immunoblots of peptides A to F, and full‐length αKlotho (Kl) by anti‐myc, anti‐Kl1 (KM2076), and anti‐Kl2 (KM2119) antibodies. The epitopes on polypeptides E and F were further refined by “peptide walking” using 21 overlapping short synthetic 20‐AA peptides by dot blot (bottom panel). The epitopes for anti‐KL1 is FRDTEALR and for anti‐KL2 is LEVQEMTD

Article Snippet: For all immunoblots, 20 μg of total protein were loaded per lane, fractionated by SDS‐PAGE, and transferred overnight onto PVDF (Thermo Scientific), which was blocked in 5% non‐fat dried milk, followed by overnight incubation at 4°C with the appropriate antibodies: anti‐KL1 (KM2076), anti‐KL2 (KM2119) (both from Trans Genic Inc.); anti‐spliced Kl1 (anti‐spE15, Custom‐generated by YenZyme), anti‐Myc (Cell Signaling); anti‐myotubularin‐related protein‐12 (anti‐MTMR12, Proteintech).

Techniques: Transfection, Plasmid Preparation, Immunocytochemistry, Western Blot, Dot Blot

Journal: FASEB BioAdvances

Article Title: In search of alternatively spliced alpha‐Klotho Kl1 protein in mouse brain

doi: 10.1096/fba.2020-00066

Figure Lengend Snippet: Immunohistochemistry of mouse brain Choroid‐plexus. Mouse brain sections were stained with anti‐KL1 (green) and anti‐spE15 (red). A, Wild type mouse brain sections was stained with anti‐spE15 (top panel) and with anti‐spE15 plus E15 peptide (bottom panel). B, (top) Wild type (top panel) and kl / kl (bottom panel) mouse brain sections was co‐stained with anti‐KL1 and anti‐spE15.

Article Snippet: For all immunoblots, 20 μg of total protein were loaded per lane, fractionated by SDS‐PAGE, and transferred overnight onto PVDF (Thermo Scientific), which was blocked in 5% non‐fat dried milk, followed by overnight incubation at 4°C with the appropriate antibodies: anti‐KL1 (KM2076), anti‐KL2 (KM2119) (both from Trans Genic Inc.); anti‐spliced Kl1 (anti‐spE15, Custom‐generated by YenZyme), anti‐Myc (Cell Signaling); anti‐myotubularin‐related protein‐12 (anti‐MTMR12, Proteintech).

Techniques: Immunohistochemistry, Staining

Journal: FASEB BioAdvances

Article Title: In search of alternatively spliced alpha‐Klotho Kl1 protein in mouse brain

doi: 10.1096/fba.2020-00066

Figure Lengend Snippet: Immunoblot (IB) and immunoprecipitation (IP) of WT mouse brain. A, Whole WT mouse brain lysate was fractionated by SDS‐PAGE and immunoblotted with the antibodies listed below. On the left panel, anti‐KL1 was used as primary antibody for detecting full‐length αKlotho and KL1 proteins in the WT mouse brain. In the left blot, no competitor peptide was added to the primary antibody solution, whilst in the middle and right blots, E15 and the KL1 peptide [FDRTEALR] were added respectively. In the right panel, anti‐spE15 was used for IB, and again from left to right, either no peptide, E15 or KL1 peptides were added as competitors in this order respectively. B, Whole WT mouse brain lysate was immunoprecipitated and the pulled‐down immune complex was resolved by SDS‐PAGE and immunoblotted with the antibodies listed left side. Anti‐spE15, rat monoclonal anti‐KL1 and anti‐KL2 were used. The most left column of three blots shows the IP using only anti‐spE15, and IB anti‐spE15, anti‐KL1 and anti‐KL2 respectively. The middle column of three blots shows the IP using anti‐KL1, and IB, anti‐spE15, anti‐KL1 and anti‐KL2 respectively. In the most right column the three blots show the IB using anti‐KL2, and IP using anti‐spE15, anti‐KL1 and anti‐KL2 respectively

Article Snippet: For all immunoblots, 20 μg of total protein were loaded per lane, fractionated by SDS‐PAGE, and transferred overnight onto PVDF (Thermo Scientific), which was blocked in 5% non‐fat dried milk, followed by overnight incubation at 4°C with the appropriate antibodies: anti‐KL1 (KM2076), anti‐KL2 (KM2119) (both from Trans Genic Inc.); anti‐spliced Kl1 (anti‐spE15, Custom‐generated by YenZyme), anti‐Myc (Cell Signaling); anti‐myotubularin‐related protein‐12 (anti‐MTMR12, Proteintech).

Techniques: Western Blot, Immunoprecipitation, SDS Page

Journal: FASEB BioAdvances

Article Title: In search of alternatively spliced alpha‐Klotho Kl1 protein in mouse brain

doi: 10.1096/fba.2020-00066

Figure Lengend Snippet: spE15 transcript is not present in mouse brain. A, Immunohistochemistry for spE15 in WT mouse choroid plexus. spE15; red, and KL1 in green. We Micro‐dissected choroid plexus from WT mouse brain were subjected to RT‐PCR using the different primers B. C, the primers (top panel), cDNA origin (middle panel) and RT‐PCR products in 1.2% agarose ethidium bromide gel (bottom panel). Lanes 2, 4, 6, 8, 10 showed strong transcript of Kl1, spKl1, and short spKl1, and very short transcripts of spKl1 and αKlotho respectively. Lanes 1, 3, 5, 7, 9, showed the transcripts in plasmids as PCR templates as positive control with. (* shows verified correct sequence.)

Article Snippet: For all immunoblots, 20 μg of total protein were loaded per lane, fractionated by SDS‐PAGE, and transferred overnight onto PVDF (Thermo Scientific), which was blocked in 5% non‐fat dried milk, followed by overnight incubation at 4°C with the appropriate antibodies: anti‐KL1 (KM2076), anti‐KL2 (KM2119) (both from Trans Genic Inc.); anti‐spliced Kl1 (anti‐spE15, Custom‐generated by YenZyme), anti‐Myc (Cell Signaling); anti‐myotubularin‐related protein‐12 (anti‐MTMR12, Proteintech).

Techniques: Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Positive Control, Sequencing