Journal: bioRxiv

Article Title: Insulin-independent glucose uptake in skeletal muscle by coupled SGLT and Na,K-ATPase transport

doi: 10.64898/2026.03.24.714065

Figure Lengend Snippet: 889 ng of DNAse-treated kidney RNA ( a ) and 927 ng of DNAse-treated muscle RNA ( b ) was reverse transcribed, the cDNA amplified by PCR, and electrophoresed on a 1% agarose gel. Lanes 1 & 20, 1 kb+ ladder; Lanes 2−7, kidney RNA with primers that target regions of Slc5a2 (primer sets 1−6, Table S3). Lane 8, kidney RNA amplified with a near full-length primer set (1896 bp of the 2070 bp coding sequence); lane 9, GAPDH (positive control); lanes 10−15, muscle RNA amplified with the same region-specific primers used for kidney; lane 16, muscle RNA amplified with the same full-length primer set used for kidney; lane 17, GAPDH ; lane 18, kidney no-RT control with GAPDH primers; lane 19, muscle no-RT control with GAPDH primers. cDNA from mouse kidney and hindlimb skeletal muscle was amplified using primer sets 1−6, 8, and GAPDH (Table S3). The same amount of cDNA was used as template for PCR of kidney and muscle. Gel is representative of 4 independent assays. The faint band in lane 15 (muscle, primer set 6 within exon 14), may be signal from the ubiquitous gene Rusf1 which contains sequences shared with exon 14 of Slc5a2 . For all primer sets, the melt curve peaks had identical melt curve/Tm values in kidney and muscle.

Article Snippet: Proteins were transferred to PDVF membranes overnight at 4 °C, 30 V. Membranes were stained with Ponceau S and washed, then incubated in TBST buffer with 2% nonfat dry milk for 1h at room temperature, followed by incubation with an SGLT2 primary antibody (ProteinTech 24654-1-AP , 1:500, 24 h; or Abcam ab37296 , 1:250, 48 h), washed 3×15 min in TBST buffer, incubated with secondary antibody (Licor IRDye @680 goat anti-Rabbit 925-68071, 1:10000)1.5 h at room temperature, washed in TBST buffer 3×15 min, and imaged (Odyssey Imager, LICORbio).

Techniques: Reverse Transcription, Amplification, Agarose Gel Electrophoresis, Sequencing, Positive Control, Control

Journal: bioRxiv

Article Title: Insulin-independent glucose uptake in skeletal muscle by coupled SGLT and Na,K-ATPase transport

doi: 10.64898/2026.03.24.714065

Figure Lengend Snippet: a – c Confocal sections of fresh-frozen mouse tibialis anterior muscle dual-labeled with primary antibodies against Na,K-ATPase-α2 (green) and SGLT2 (red), and merged image. Scale bar 50 μm. d – f Longitudinal section of paraformaldehyde-fixed EDL fibers dual-labeled with MAP17 (green) and SGLT2 (red) antibodies, and merged image. Scale bar 13.5 μm. g – i cross-sections of mouse soleus muscle dual-labeled with myosin type I (green) and SGLT2 (red) antibodies, and merged image. Scale bar 145 μm. j – l Human vastus lateralis muscle dual-labeled with primary antibodies against MAP17 (green) and SGL2 (red), and merged image. Scale bar 145 μm. 10 μm sections. Images are representative of a minimum of three sections from each tissue sample. Negative controls included the secondary antibody and no primary antibody, and showed no signal for all sections (data not shown). Western Blot of mouse hindlimb skeletal muscle lysate (140 μg per lane) and mouse whole kidney lysate (20 μg per lane), labeled with the same SGLT2 antibody. n Western Blot of plasma membranes purified from mouse hindlimb skeletal muscle labeled with the same antibody. Muscle and kidney lysates were solubilized and denatured using a standard loading buffer with 2.5% SDS, 50 μM β-ME, and heated at 37 C for 30 min. o, p Western blots of purified plasma membranes from mouse hindlimb skeletal muscle (40 μg per lane) labeled with the same antibody used for IHC (PT) or an independent SGLT2 antibody (Abcam). The same muscle preparation was treated identically up to the antibody incubation step. This membrane preparation was solubilized and denatured more aggressively using 5% SDS, 100 mM DTT and heating at 65C for 15 min. q, r positive controls: human kidney cortical epithelial cells labeled with PT or Abcam antibodies. Western Blot images are representative of 3-4 blots obtained using 3 independent kidney and muscle samples. Control images without primary antibody and after Ponceau staining are provided in . Western blot images are representative of 4 blots obtained using 3 independent kidney and muscle samples.

Article Snippet: Proteins were transferred to PDVF membranes overnight at 4 °C, 30 V. Membranes were stained with Ponceau S and washed, then incubated in TBST buffer with 2% nonfat dry milk for 1h at room temperature, followed by incubation with an SGLT2 primary antibody (ProteinTech 24654-1-AP , 1:500, 24 h; or Abcam ab37296 , 1:250, 48 h), washed 3×15 min in TBST buffer, incubated with secondary antibody (Licor IRDye @680 goat anti-Rabbit 925-68071, 1:10000)1.5 h at room temperature, washed in TBST buffer 3×15 min, and imaged (Odyssey Imager, LICORbio).

Techniques: Labeling, Western Blot, Clinical Proteomics, Purification, Incubation, Membrane, Control, Staining

Journal: bioRxiv

Article Title: Insulin-independent glucose uptake in skeletal muscle by coupled SGLT and Na,K-ATPase transport

doi: 10.64898/2026.03.24.714065

Figure Lengend Snippet:

Article Snippet: Proteins were transferred to PDVF membranes overnight at 4 °C, 30 V. Membranes were stained with Ponceau S and washed, then incubated in TBST buffer with 2% nonfat dry milk for 1h at room temperature, followed by incubation with an SGLT2 primary antibody (ProteinTech 24654-1-AP , 1:500, 24 h; or Abcam ab37296 , 1:250, 48 h), washed 3×15 min in TBST buffer, incubated with secondary antibody (Licor IRDye @680 goat anti-Rabbit 925-68071, 1:10000)1.5 h at room temperature, washed in TBST buffer 3×15 min, and imaged (Odyssey Imager, LICORbio).

Techniques:

Journal: Experimental & Molecular Medicine

Article Title: Deficiency of exocyst complex component Exoc5 exacerbates the progression of kidney fibrosis

doi: 10.1038/s12276-026-01649-8

Figure Lengend Snippet: a The kidney sections were subjected to immunofluorescence staining using antibodies against Exoc5 (green) and AQP1 (a marker of proximal tubule cells, red). DAPI staining was used to visualize cell nuclei. b , c The Exoc5 expression in the kidneys of PT–Exoc5 WT and PT–Exoc5 KO mice was analyzed by western blot. GAPDH was used as the loading control. Densities of bands were quantified using ImageJ software ( n = 3). d Representative images of PT–Exoc5 WT and PT–Exoc5 KO mice. e Body weights of PT–Exoc5 WT and PT–Exoc5 KO mice. f Representative images of kidneys of PT–Exoc5 WT and PT–Exoc5 KO mice. Kidney sections were subjected to PAS staining. g Kidney weights. h – m PCr ( h ) and BUN ( i ) concentrations, CrCl ( j ), urine osmolality (Osmol) ( k ), urine Na + /K + ratio ( l ) and urine glucose concentrations ( m ) were measured. n The kidney sections were subjected to immunohistochemical staining using antibodies against AQP1, Na + /K + ATPase and SGLT2. Results are expressed as the mean ± s.e.m. ( n = 4–6). Blood and urine were collected from PT–Exoc5 WT and PT–Exoc5 KO mice as described in . * P < 0.05. NS, not significant.

Article Snippet: Immunohistochemical staining was performed using antibodies against AQP1 (cat. no. ab168387, Abcam), Na + /K + ATPase (cat. no. ab76020, Abcam), SGLT2 (cat. no. 24654-1-AP, Proteintech) and F4/80 (cat. no. MCA497GA, Bio-Rad Laboratories) antibodies.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Western Blot, Control, Software, Immunohistochemical staining

Journal: Experimental & Molecular Medicine

Article Title: Deficiency of exocyst complex component Exoc5 exacerbates the progression of kidney fibrosis

doi: 10.1038/s12276-026-01649-8

Figure Lengend Snippet: a , d Western blot of p-Smad3, Smad3 and β-catenin ( a ) and α-SMA and vimentin ( d ). b , c , e , f Graphs of p-Smad3/Smad3 ( b ), β-catenin ( c ), α-SMA ( e ) and vimentin ( f ) expression in the kidney tissue. GAPDH was used as the loading control, and densities of bands were quantified using ImageJ software. g , j , k , n Kidney sections were subjected to immunostaining using antibodies against α-SMA (red), vimentin (green in g and red in j ), and Exoc5 (green), AQP1 (red in g and brown in n ), E-cadherin (green), ZO-1 (green), and SGLT2 (brown). DAPI staining (blue) was used to visualize cell nuclei. The white arrowheads indicate vimentin positivity in tubular cells, and the black arrowheads indicate cells with apical loss of AQP1 or SGLT2 expression. In j , adjacent serial kidney sections are immunostained for Exoc5 (green) and vimentin (red). The filled arrowheads indicate vimentin-expressing tubular cells. The open arrowheads indicate interstitial cells co-expressing Exoc5 and vimentin. h , i , l , m , o , p The quantification was performed by measuring the area of α-SMA ( h ), vimentin ( i ), E-cadherin ( l ), ZO-1 ( m ), AQP1 ( o ) and SGLT2 positivity ( p ). PT–Exoc5 WT and PT–Exoc5 KO mice were subjected to either UUO or sham surgery, and kidneys were collected 7 days following surgery. Results are expressed as the mean ± s.e.m. ( n = 4–6). * P < 0.05. NS, not significant.

Article Snippet: Immunohistochemical staining was performed using antibodies against AQP1 (cat. no. ab168387, Abcam), Na + /K + ATPase (cat. no. ab76020, Abcam), SGLT2 (cat. no. 24654-1-AP, Proteintech) and F4/80 (cat. no. MCA497GA, Bio-Rad Laboratories) antibodies.

Techniques: Western Blot, Expressing, Control, Software, Immunostaining, Staining