primary antibodies against ha  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Effects of proximal tubule-specific ATRAP enhancement on hypertension in a remnant kidney chronic kidney disease model of mice

doi: 10.1038/s41598-025-12168-3

Figure Lengend Snippet: Expression and localization of angiotensin II type 1 receptor-associated protein (ATRAP) in proximal tubule-specific ATRAP transgenic (PT-Tg) and littermate-control (LC) mice. (a) Representative western blots and quantitative analysis of ATRAP in the kidney of littermate-control (LC) and proximal tubule-specific ATRAP transgenic (PT-Tg) mice. Values are expressed as mean ± SEM (n = 5 in each group). *** P < 0.001 versus LC, unpaired t -test. (b) Renal cortical sections showing ATRAP expression in renal tubules detected by anti-ATRAP antibody (left panels). Consecutive sections were stained with a monoclonal antibody against megalin (middle panels), a specific marker of proximal tubule (PT), and a monoclonal antibody against calbindin-D (right panels), a specific marker of distal convoluted tubule (DT) and connecting tubule. Original magnification: × 400. (c, d, e) Quantitative analysis of calbindin-D, megalin and ATRAP mRNA expression in Glo, PTs, and DTs of the renal cortex identified by the LMD method. Values are normalized relative to 18S rRNA control levels. Values are expressed as mean ± SEM (n = 4 mice in mix). ATRAP, Angiotensin II type 1 receptor-associated protein; LC, littermate-control; PT-Tg, proximal tubule-specific ATRAP transgenic; Glo, glomerulus; PT, proximal tubule; DT, distal tubule; LMD, Laser‐capture microdissection.

Article Snippet: Samples were run on a 5–20% polyacrylamide gel, transferred to PVDF membranes, blocked, and probed with specific primary antibodies against ATRAP (diluted 1:2000, developed in our laboratory as previously described – ), HA (diluted 3:2000, A190-108A-2; Bethyl Laboratories, Montgomery, TX, USA) and GAPDH (diluted 1:2000, GTX100118; GeneTex, Irvine, CA, USA).

Techniques: Expressing, Transgenic Assay, Control, Western Blot, Staining, Marker, Laser Capture Microdissection

Journal: PLOS Biology

Article Title: SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila

doi: 10.1371/journal.pbio.3002941

Figure Lengend Snippet: (A) SLMO localizes to mitochondria. Muscle and testis tissues of ubi-slmo-GFP flies were dissected and stained with GFP (green) and TOM20 (red). The Arrow head indicates aggregated mitochondria within the Nebenkern at the onion stage. Asterisks are used to mark elongated mitochondria in mature spermatozoa. Scale bar, 10 μm. (B) Cell lysates from S2 cells transfected with SLMO-GFP were analyzed using the protease K protection assay. Without Triton X-100, SLMO and CoIV were resistant to protease K treatment, whereas TOM20 was sensitive. (C) S2 cells expressing SLMO-sp1-10 and SDHC(1–94)-sp11 or SDHC(1–139)-sp11 were imaged using confocal fluorescent microscopy. TOM20 (red) was stained to visualize mitochondria. The scale bar represents 10 μm. (D) Super-resolution SIM microscopy localizes SLMO to the IBM. Muscle tissues from slmo-HA flies, ubi-MICU1-sp1-10 (could be stained by antibody anti-GFP), and ubi-SDHC(1–94)-GFP flies were penetrated by Triton X-100 or 5 μg/ml digitonin, followed by staining for SLMO-HA and MICU1-sp1-10 (left, red), TOM20 (left, green), and GFP (right, red). Scale bar, 10 μm. The OMM (green arrowhead) and IBS (red) regions of IMS of mitochondria are illustrated. (E) Staining of mitochondria from muscles of ubi-SDHC(1–94)-mCherry by TOM20 (green) and mCherry (red) antibodies. The OMM (white arrow) and CM (yellow arrow head) of mitochondria are illustrated. The tissues were penetrated by Triton X-100. Scale bar, 10 μm. CM, cristae membrane; IBS, inner boundary space; IMS, inner membrane space.

Article Snippet: Cells on a coverslip were incubated with primary antibodies against Tom20 (rat, 1:100) [ ], GFP (rabbit, 1:200, Invitrogen), Cnx99 (mouse, 1:100, Developmental Studies Hybridoma Bank), or HA (mouse, 1:200, Santa Cruz).

Techniques: Staining, Transfection, Expressing, Microscopy, Muscles, Membrane

Journal: PLOS Biology

Article Title: SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila

doi: 10.1371/journal.pbio.3002941

Figure Lengend Snippet: (A) SLMO transfer PS between liposomes. A schematic diagram of the lipid transfer assay is shown on the left, in which donor liposomes with NBD-PS or NBD-PE, and acceptor liposomes were incubated with MBP-SLMO. Time courses of normalized fluorescence signals from liposome mixtures containing NBD-PS or NBD-PE in the donor liposomes with SLMO or BSA are plotted. (B) Design of mutant SLMO in PS harboring. From MD calculation, Thr93 and Asn150 interact with PS, thus mutating both Thr93 and Asn150 to Ala may prevent PS binding in the pocket. (C) SLMO T93A and SLMO N150A abolish the PS transporting ability. Time courses of normalized fluorescence signals from liposome mixtures containing NBD-PS in the donor liposomes with SLMO, SLMO T93A , or SLMO N150A are shown. (D, E) SLMO T93A and SLMO N150A are not able to replace SLMO in promoting mitochondrial organization and cellular function. (D) Muscles sections of control ( MHC-gal4/UAS-GFP RNAi ), slmo RNAi1 ( MHC-gal4/UAS-slmo RNAi1 ), slmo RNAi1 +EGFP ( MHC-gal4/+;UAS-slmo RNAi1 /UAS-GFP ), slmo RNAi1 +slmo R1 ( MHC-gal4/+;UAS-slmo RNAi1 /DA-slmo R1 ), slmo RNAi1 +slmo T93A ( MHC-gal4/+;UAS-slmo RNAi1 /DA-slmo T93A ), and slmo RNAi1 +slmo N150A ( MHC-gal4/+;UAS-slmo RNAi1 /DA-slmo N150A ) flies. M, Mitochondria; F, myofibrils. Scale bar, 1 μm. (E) Quantification of ERG amplitudes and off-transients of control ( GMR-gal4/UAS-GFP RNAi ), slmo RNAi1 ( GMR-gal4/UAS-slmo RNAi1 ), slmo RNAi1 +EGFP ( GMR-gal4/+;UAS-slmo RNAi1 /UAS-GFP ), slmo RNAi1 +slmo T93A ( GMR-gal4/+;UAS-slmo RNAi1 /UAS-slmo T93A ), slmo RNAi1 +slmo N150A ( GMR-gal4/+;UAS-slmo RNAi1 /DA-slmo N150A ), and slmo RNAi1 +slmo R1 ( GMR-gal4/+;UAS-slmo RNAi1 /DA-slmo R1 ) flies. ERG from at least 10 flies was used, and significant differences were determined using the unpaired t test. DA-slmo R1 , DA-slmo T93A , and DA-slmo N150A were designed as slmo RNAi1 resistant. (F) Lipidomic analysis of mitochondrial PE and PS levels of control ( MHC-gal4/GFP RNAi ), pisd RNAi ( MHC-gal4/UAS-pisd RNAi ), slmo RNAi1 ( MHC-gal4/UAS-slmo RNAi1 ), and slmo RNAi + slmo R1 ( MHC-gal4/+;UAS-slmo RNAi /UAS-slmo R1 ) muscles. Mitochondria were isolated from 10 dissected thoraxes per assay, and 4 replicates were quantified. (G) Knocking down slmo in S2 cells increased PS levels in the OMM. Live images of cells transfected with Tom20-PSS (red), GFP-LactC1C2 (green), and dsRNA of BFP (control) or slmo ( slmo RNAi ). Scale bar, 10 μm. Manders’ Colocalization Coefficients (MCC) of GFP-LactC1C2 and Tom20-RFP of S2 cells transfected with BFP RNAi , TOM20-PSS, slmo RNAi pss RNAi , or pisd RNAi were quantified by ImageJ. The data underlying the graphs shown in the figure can be found in . ERG, electroretinogram; MD, molecular dynamics; OMM, outer mitochondrial membrane; PE, phosphatidylethanolamine; PS, phosphatidylserine.

Article Snippet: Cells on a coverslip were incubated with primary antibodies against Tom20 (rat, 1:100) [ ], GFP (rabbit, 1:200, Invitrogen), Cnx99 (mouse, 1:100, Developmental Studies Hybridoma Bank), or HA (mouse, 1:200, Santa Cruz).

Techniques: Liposomes, Incubation, Fluorescence, Mutagenesis, Binding Assay, Cell Function Assay, Muscles, Control, Isolation, Transfection, Membrane

Journal: PLOS Biology

Article Title: SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila

doi: 10.1371/journal.pbio.3002941

Figure Lengend Snippet: The dtriap1 and dtriap2 loci and mutation sites associated with dtriap1/2 KO . The asterisk indicates a stop codon. (B–D) Knocking out dtriap1 and dtriap2 does not affect (B) mitochondrial morphology, (C) neuronal function, or (D) PE/PS level of mitochondria. (B) TEM sections of muscles from wild-type ( nos-Cas9 ) and dtriap1/2 KO flies show similar mitochondrial size. M, mitochondria; F, myofibrils. Scale bar, 2 μm. (C) ERG responses of wild-type and dtriap1/2 KO flies are shown. (D) Lipidomic analysis of mitochondrial phospholipid levels of control ( nos-Cas9 ), dtriap1/2 ko muscles. Individual phospholipid levels were calculated in molar fractions of total phospholipids. Mitochondria were isolated from 10 dissected thoraxes per genotype, and 4 replicates were quantified. (E) co-expression of dTRIAP1 and SLMO did not affect mitochondria size. Quantification of mitochondria size in indirect flight muscles from control ( MHC-gal4/UAS-RFP ), dtriap2 ( MHC-gal4/UAS-dtriap2 ), dtriap2+slmo ( MHC-gal4/+;UAS-slmo/UAS-dtriap2 ), slmo ( MHC-gal4/UAS-slmo ), dtriap1 ( MHC-gal4/UAS-dtriap1 ), and dtriap1+slmo ( MHC-gal4/+;UAS-slmo/UAS-dtriap1 ) flies. All flies were raised for 5 days under 12 h-light/12 h-dark cycles. (F) Knockout of dtriap1 /2 failed to affect mitochondrial morphology in slmo RNAi flies. Quantification of mitochondria size in flight muscles from control ( MHC-gal4/UAS-GFP ), dtriap1/2 RNAi ( MHC-gal4/UAS-dtriap1/2 RNAi ), slmo RNAi2 ( MHC-gal4/+;UAS-slmo RNAi2 /+ ), and dtriap1/2 RNAi +slmo RNAi2 ( MHC-gal4/UAS-dtriap1/2 RNAi ;UAS-slmo RNAi2 ) flies. Six samples were analyzed for each phenotype. (G–I) dTRIAP1 and dTRIAP2 localize to the OMM. (G) dTRIAP1 is located to mitochondria. S2 cells were transfected with untagged and MYC-tagged dTRIAP1 (dTRIAP1-myc, bottom) and labeled using antibodies against dTRIAP1 (top, green), MYC (bottom, green), and TOM20 (red). Scale bar, 10 μm. (H) S2 cells co-expressing dTRIAP1-sp1-10 or dTRIAP2-sp1-10 with TOM20-sp11, SDHC(1–94)-sp11, or SDHC(1–139)-sp11 were imaged for GFP fluorescence (green); TOM20 (red) was used to visualize mitochondria. Scale bar, 10 μm. (I) Protease K protection assay of dTRIAP1 in S2 cells transfected with dTRIAP1. The data underlying the graphs shown in the figure can be found in . ERG, electroretinogram; OMM, outer mitochondrial membrane; PE, phosphatidylethanolamine; PS, phosphatidylserine; TEM, transmission electron microscopy.

Article Snippet: Cells on a coverslip were incubated with primary antibodies against Tom20 (rat, 1:100) [ ], GFP (rabbit, 1:200, Invitrogen), Cnx99 (mouse, 1:100, Developmental Studies Hybridoma Bank), or HA (mouse, 1:200, Santa Cruz).

Techniques: Mutagenesis, Muscles, Control, Isolation, Expressing, Knock-Out, Transfection, Labeling, Fluorescence, Membrane, Transmission Assay, Electron Microscopy

Journal: PLOS Biology

Article Title: SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila

doi: 10.1371/journal.pbio.3002941

Figure Lengend Snippet: (A) Expressing SLMO2 restored ERG responses of slmo mutant flies. Five-day-old control ( GMR-Gal4/UAS-EGFP RNAi ), slmo RNAi1 ( GMR-Gal4/UAS-slmo RNAi1 ), slmo RNAi1 +EGFP ( GMR-Gal4/+;UAS-slmo RNAi1 /UAS-EGFP ), slmo RNAi +PRELID1 ( GMR-Gal4/+;UAS-slmo RNAi1 /UAS-PRELID1 ), slmo RNAi1 +SLMO1 ( GMR-Gal4/+;UAS-slmo RNAi1 /UAS-SLMO1 ), and slmo RNAi1 +SLMO2 ( GMR-Gal4/+;UAS-slmo RNAi1 /UAS-SLMO2 ) flies were recorded. (B–D) SLMO2 localized to the IMS of mitochondria. Hela cells expressing SLMO2-GFP or PRELID1-GFP were penetrated by (B) triton or (C) digitonin, and GFP was stained (red, in C) or directly observed (green, in C). TOM20 (red, in B) are stained as markers for mitochondria. (D) Hela cells co-expressing SLMO2-sp1-10 (white) with SDHC(1–94)-mCherry-sp11 or SDHC(1–139)-mCherry-sp11 were directly imaged for GFP fluorescence (green) and mCherry fluorescence (red). Scale bar, 10 μm. (E) TEM images show that knocking down Slmo2 in Hela cells caused mitochondrial fragmentation and cristae loss. Mitochondria were labeled as “m.” Slmo2 was knocked down in Hela cells by transfecting them with pLKO vectors expressing Slmo2 RNAi1 and Slmo2 RNAi2 and were rescued by expressing wild-type SLMO2 with corresponding shRNA -resistant DNA sequence changes. (F) Mitochondrial size and cristae density were quantified from the mitochondria of more than 20 cells in each group. (G) OCRs in Hela cells transfected with control, Slmo2 RNAi1 , Slmo2 RNAi2 , Slmo2 RNAi1 +SLMO2, and Slmo2 RNAi2 +SLMO2. ATP production and maximal respiration of the indicated genotype were calculated by normalization of OCR levels to ATP levels. The data underlying the graphs shown in the figure can be found in . ATP, adenosine triphosphate; ERG, electroretinogram; IMS, inner membrane space; OCR, oxygen consumption rate; TEM, transmission electron microscopy.

Article Snippet: Cells on a coverslip were incubated with primary antibodies against Tom20 (rat, 1:100) [ ], GFP (rabbit, 1:200, Invitrogen), Cnx99 (mouse, 1:100, Developmental Studies Hybridoma Bank), or HA (mouse, 1:200, Santa Cruz).

Techniques: Expressing, Mutagenesis, Control, Staining, Fluorescence, Labeling, shRNA, Sequencing, Transfection, Membrane, Transmission Assay, Electron Microscopy